DESC:FIELD OF THE INVENTION:
The present invention belongs to the field of biotechnology, in particular the field of biochemical composition and process of preparation thereof. The present invention more particularly relates to composition of ingredients as a serum alternative from an animal origin free source and development of serum alternative from the same. More particularly, the present invention further relates to the specific composition as a serum alternative and application thereof in animal cell culture, biopharmaceutical ingredients in antibody and vaccine preparation, formulation of final drug components and other translation research work like stem cell research.

BACK GROUND OF THE INVENTION:
Serum is the amber-colored blood fraction remaining after the natural coagulation of the blood; it is typically further refined via centrifugation, which serves to remove remaining blood cells, coagulation fibrinogens, and low-solubility proteins.

Fetal bovine serum is derived from the blood of a cow foetus, which is drawn via a closed system of collection. Notably, FBS is the most widely used serum supplement for in vitro cell culture (for eukaryotic cells).

Fetal bovine serum (FBS), also known as fetal calf serum, is used extensively by both academic biology and industrial researchers as a supplement to basal growth medium in cell culture applications. It is a rich source of proteins and growth factors that support cell growth in culture.

Gene overexpression is the process which leads to the abundant target protein expression subsequently. The process may be in the cell where the gene is originally located or in other expression systems. The fundamental principle is to add regulatory elements to the upstream of the target gene through artificial construction, so that genes can be transcribed and translated efficiently under controlled conditions.

Successful production of protein of interest has been accomplished in both prokaryotic and eukaryotic hosts. The most prominent examples are fungi such as Escherichia coli, yeasts such as Saccharomyces cerevisiae, Pichia pastoris or Hansenula polymorpha, and Aspergillus awamori; or filamentous fungi such as Trichoderma reesei, or mammalian cells such as CHO cells. Yields of some proteins are easily achieved at high rates, but many other proteins are only produced at relatively low levels.

Recombinant proteins produced by the strain engineering used for replacement of fetal bovine serum which is a major problem for cell culture industry. The following problems are solved by serum replacement proteins or alternative serum.

Significant batch-to-batch variation exists because serum is a complex mixture of an undefined composition of biomolecules

Majority of proteins and other components are not defined and characterized due to which there is unexpected and undesired outcomes.

Risk of contamination by other proteins or pathogens is always problematic for biological drug or vaccine productions

Fetal bovine serum (FBS) is recovered from cow which is not a limited source and shortage of FBS in future is certain due to expansion of biopharmaceutical companies working for cell culture-based biomolecules.
To improve the secretion of recombinant proteins, one strategy is to develop a system of overexpressing of animal origin free of human transferrin, human albumin, fibronectin, Vitronectin, Human TGF beta and FGF in yeast or bacterial host system for development of serum alternative used in animal cell culture.
Fetal bovine serum used as a supplement in culture media primarily aids in supplying hormone factors for cell proliferation and growth; Providing nutrients, trace elements, transport proteins, adherence, and extension factors; Cultivating a suitable environment for growth with stabilizing and detoxifying factors; Stimulating cell differentiation

Academic researchers as well as those in pharmaceutical and biotechnological industries have relied on the valuable properties of FBS for many years. Specific applications of FBS in research and industry include but are not limited to Animal diagnostics, Cloning, Cryopreservation, Stem-cell research, In vitro fertilization, Biopharmaceuticals, Cell and gene therapy production, Immunotherapy, Synthetic protein production, Viral vector production, Vaccine production.

The primary application or use of FBS is as a growth supplement for in vitro cell culture, and it is typically added to basal cell culture media at a concentration of 5–10%. While other animal sera (e.g., horse, rabbit, goat, porcine, etc.) are also available and utilized for cell culture, fetal bovine serum remains the most universally employed.

The unique biological makeup of FBS promotes rapid cell growth, thus making it a product that yields a high efficacy. In addition, fetal bovine serum contains a sparse amount of gamma globulin, higher levels of growth factors, and fewer complement proteins than both calf and adult bovine serum. This makes FBS ideal for propagating cell growth while also decreasing the possibility of mammalian cells binding or lysing in the culture, rationalizing the preference of FBS over other types of bovine sera. Furthermore, FBS contains low levels of antibodies and other growth-inhibiting components.

In addition to providing cells with many growth-enhancing factors (growth factors, hormones, nutrients, etc.), FBS can also protect cells from harmful disruptions, including large pH shifts, proteases, toxic agents, shear forces, agents that would typically break up monolayers of adherent cells (FBS acts to inactivate these agents. Therefore, when FBS is used, cell growth is typically: rapid, consistent and reproducible, lacking in undesirable changes in differentiation, not hampered by the introduction of detrimental contaminants.

While other animal sera, such as horse serum, may be good for certain types of applications, FBS is more universal and can function across a variety of applications, streamlining supply needs.

Although researchers assume that all FBS products are of the same quality, there are several key factors that should be monitored and considered when sourcing FBS for the lab, including: supply continuity, lot-to-lot consistency, reproducible results, price fluctuation, product integrity. Furthermore, researchers rely on FBS that is sterile, filtered, and free of mycoplasma.

Moreover, the world is moving towards environmentally friendly, animal friendly and ethical methods of biotechnological research. Hence, there is an increasing need for animal-serum free media which ensures continuous supply, and consistent quality of the product. Furthermore, the animal-serum free media is prepared through precision fermentation which improves the scalability and economically viable solution.

The present invention has found that specific composition of ingredients used in present invention and preparation of alternative serum of the present invention is found to be useful in animal cell culture, biopharmaceutical ingredients, formulation of final drug components and other translation research work like stem cell research and suitably overcomes all the problem mentioned in prior arts.

Further the biochemical composition of the present invention is found to be easy to use. Further present invention avoid the dependency over the foetal bovine serum.

SUMMARY OF THE INVENTION:
The principal aspect of the present invention is to formulate a biochemical composition as an alternative to fetal bovine serum (FBS) from animal-origin free source.

Another aspect of the present invention is to provide biochemical composition as a serum alternative from animal origin free source comprising different fractions of purified algal extract, FGF2, TGF Beta, VEGF, EGF, Transferrin, Albumin, Insulin, Selenium, Fetuin, Growth factors, Others salts, others attachment factors selected from vitronectin , fibronectin or combination thereof.

In further aspect of the present invention is to provide the biochemical composition of animal-origin free FBS comprising FGF2 present in an amount of 2-100 ng/mL; VEGF present in an amount of 0.1-50 ng/mL; EGF present in an amount of 0.5-20 ng/mL; PDGF present in an amount of 1-50 ng/mL; and TGF beta present in an amount of 1-10 ng/mL.

Yet another aspect of the present invention is to provide biochemical composition of animal-origin free FBS comprising Albumin present in an amount of 0.1% to 2.0% w/v; Transferrin present in an amount of 1.0-12.0% w/v; Fetuin-A present in an amount of 0.25-2.0% w/v; Growth factors present in an amount of 0.5%-5 % w/v; Others salts present in an amount of 10-12% w/v; and other attachment factors as vitronectin, fibronectin are present in an amount of 4-6% w/v.

In further aspect of the present invention is to provide essential growth factors to cells line that are inoculated, to help aid cell proliferation, cell viability and enhanced cell differentiation.

Another aspect of the present invention provides biochemical composition as a serum alternative from animal origin free source and process of preparation of the said composition and formulation thereof.

Further aspect of the present invention is to provide biochemical composition as a serum alternative from animal origin free source and application thereof in animal cell culture, biopharmaceutical ingredients in antibody and vaccine preparation, formulation of final drug components and other translation research work like stem cell research.

Therefore an aspect of the present invention provides a biochemical composition as an alternative for a bovine serum comprising all the essential elements present in a predetermined quantity provided products are of the same quality, and avoids dependency over the limited source of bovine serum and supply continuity, provide consistency at each repeated application, provides a consistent reproducible results, overcomes the problem associated with a price fluctuation. Furthermore, composition of the present invention is sterile, filtered, and free of mycoplasma.

DETAILED DESCRIPTION OF THE INVENTION:
Serum is the fluid and solvent component of blood which does not play a role in clotting. It may be defined as blood plasma without the clotting factors, or as blood with all cells and clotting factors removed. Serum contains all proteins except clotting factors (involved in blood clotting), including all electrolytes, antibodies, antigens, hormones; and any exogenous substances (e.g., drugs, microorganisms). Serum also does not contain all the formed elements of blood, which include blood cells, white blood cells (leukocytes, lymphocytes), red blood cells (erythrocytes), and platelets.

The study of serum is serology. Serum is used in numerous diagnostic tests as well as blood typing. Measuring the concentration of various molecules can be useful for many applications, such as determining the therapeutic index of a drug candidate in a clinical trial.

“Over expression” as mentioned herein means that process involving the conversion of from limited source to make a desired (protein) in sufficient quantity by suitable method for further study.

“Recombinant” as mentioned herein means production of DNA, proteins, cells, or organisms that are made by combining genetic material from two different sources from recombinant DNA technology.

Fetal bovine serum (FBS) is rich in growth factors and is frequently added to growth media used for eukaryotic cell culture. A combination of FBS and the cytokine leukemia inhibitory factor was originally used to maintain embryonic stem cells, but concerns about batch-to-batch variations in FBS have led to the development of serum substitutes.

Therefore as aspect of the present invention provides a biochemical composition as an alternative for a bovine serum comprising all the essential elements present in a very specific quantity providing products are of the same quality, and avoids dependency over the limited source of bovine serum and supply continuity, provide consistency at each repeated application, provides a consistent reproducible results, overcomes the problem associated with a price fluctuation, product integrity. Furthermore, composition of the present invention is sterile, filtered, and free of mycoplasma.

Therefore the principal object of the present invention is to provide a biochemical composition as a serum alternative from animal origin free source.

Another objective of the present invention is to provide biochemical composition as a serum alternative from animal origin free source comprising different fractions of purified algal extract, FGF2, TGF Beta, Transferrin, Albumin, Insulin, Selenium, Fetuin, Growth factors, Others salts, others attachment factors selected from vitronectin, fibronectin or combination thereof.

In further aspect of the present invention is to provide the biochemical composition of animal-origin free FBS comprising FGF 2 present in an amount of 2-100 ng per liter; and TGF beta present in an amount of 1-10 ng per liter.

Yet another aspect of the present invention is to provide biochemical composition of animal-origin free FBS comprising Albumin present in an amount of 0.1-2.0 %; Transferrin present in an amount of 5-12%; Fetuin-A present in an amount of 6-10%; Growth factors present in an amount of 0.5%-5 %; Others salts present in an amount of 10-12%; and other attachment factors as vitronectin, fibronectin are present in an amount of 4-6%.

Another aspect of the present invention provides biochemical composition as a serum alternative from animal origin free source and process of preparation of the said composition and formulation thereof.

Further aspect of the present invention is to provide biochemical composition as a serum alternative from animal origin free source and application thereof in animal cell culture, biopharmaceutical ingredients in antibody and vaccine preparation, formulation of final drug components and other translation research work like stem cell research.

Accordingly, in an another aspect, of the process of the overexpression of animal origin free protein for the present invention is applicable in animal cell culture, biopharmaceutical ingredients, formulation of final drug components and other translation research work like stem cell research.

Minimal Essential Medium:
Minimum Essential Medium (MEM), developed by Harry Eagle, is one of the most widely used synthetic cell culture media. Early attempts to cultivate normal mammalian fibroblasts and certain subtypes of HeLa cells revealed that they had specific nutritional requirements that could not be met by Eagle's Basal Medium (BME). Subsequent studies using these and other cells in culture indicated that additions to BME could be made to aid the growth of a wider variety of fastidious cells.

MEM, which incorporates these modifications, includes higher concentrations of amino acids so it more closely approximates the protein composition of mammalian cells. Glucose is typically present in MEM at a concentration of around 4-5 mM. MEM also contains other essential nutrients such as vitamins and minerals to support cell growth, but does not contain certain key components such as proteins, lipids, and growth factors. It may require supplementation with fetal bovine serum (FBS) to support cell growth and viability.

MEM is formulated to be similar to the body's natural fluids and has been used for cultivation of a wide variety of cells grown in monolayers. Optional supplementation of MEM that incorporate either Hanks' or Earle's salts has broadened the usefulness of this medium. The formulation has been further modified by the optional elimination of calcium, which aids the growth of cells in suspension.

MEM is an important tool in many areas of scientific research, including tissue engineering, drug discovery, and the study of cell biology. It is widely used in cell culture because it is relatively inexpensive and easy to use, and it has been shown to support the growth of a variety of cell types.

F12 cell culture medium:
F-12 Nutrient Mix was designed by Ham in 1969 based on Ham's F-10 Nutrient Mixture, initially for serum-free culture of CHO cells. Ham's F-12 is often used as a basic culture medium for serum-free culture, and is especially suitable for single-cell culture and clonal culture at low serum content, and is also widely used in the culture of cancer cells and primary cells after adding serum, such as rat liver cells, rat prostate epithelial cells, chondrocytes, rat myoblasts, chicken embryonic cells, etc. In addition, Ham's F-12 mixed with DMEM and other volumes results in a more nutrient-rich and widely used DMEM/F12 medium.

This product contains amino acids, vitamins, inorganic salts and other ingredients required for cell culture, but does not contain proteins, lipids or any growth factors, so the product needs to be used with serum or serum-free additives.

Compared to other basal media, F-12 contains a wider variety of components, including zinc, putrescine, hypoxanthine, and thymidine. F-12 contains no proteins or growth factors. Therefore, F-12 requires supplementation, commonly with 10% Fetal Bovine Serum (FBS). F-12 uses a sodium bicarbonate buffer system (1.176 g/L), and therefore requires a 5–10% CO2 environment to maintain physiological pH.

Fibroblast Growth Factor 2:
Fibroblast Growth Factor 2 (FGF2, also known as basic FGF, bFGF) is a pleiotropic regulator of proliferation, differentiation, migration, and survival in a variety of cell types and is an essential component of media for human pluripotent stem cells (PSC) cultivation because it helps maintain the cells in the pluripotent state. Pluripotency is the ability of cells to undergo indefinite self-renewal and differentiate into all cell types of the human body. This property makes cells valuable for studying embryogenesis, for drug discovery, and for cell-based therapies. Other important biological activities of FGF2 that cover medicinal use include promotion of angiogenesis, promotion of wound healing, promotion of chondrogenesis or osteogenesis, and promotion of neurogenesis. However, low stability and short half-life of the wild-type FGF2 is not practical for several applications, including cultivation of PSC.

Like other FGF family members, FGF2 possesses broad mitogenic and cell survival activities, and is involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion.
In normal tissue, FGF2 is present in basement membranes and in the subendothelial extracellular matrix of blood vessels. It stays membrane-bound as long as there is no signal peptide.

It has been found that during both wound healing of normal tissues and tumor development, the action of heparan sulfate-degrading enzymes activates FGF2, thus mediating the formation of new blood vessels, a process known as angiogenesis.

In addition, FGF2 is synthesized and secreted by human adipocytes and the concentration of FGF2 correlates with the BMI in blood samples. It was also shown to act on preosteoblasts – in the form of an increased proliferation – after binding to fibroblast growth factor receptor 1 and activating phosphoinositide 3-kinase.

FGF2 has been shown in preliminary animal studies to protect the heart from injury associated with a heart attack, reducing tissue death and promoting improved function after reperfusion.

Recent evidence has shown that low levels of FGF2 play a key role in the incidence of excessive anxiety.

Additionally, FGF2 is a critical component of human embryonic stem cell culture medium; the growth factor is necessary for the cells to remain in an undifferentiated state, although the mechanisms by which it does this are poorly defined. It has been demonstrated to induce gremlin expression which in turn is known to inhibit the induction of differentiation by bone morphogenetic proteins. It is necessary in mouse-feeder cell dependent culture systems, as well as in feeder and serum-free culture systems. FGF2, in conjunction with BMP4, promote differentiation of stem cells to mesodermal lineages. After differentiation, BMP4 and FGF2 treated cells generally produce higher amounts of osteogenic and chondrogenic differentiation than untreated stem cells. However, a low concentration of FGF2 may exert an inhibitory effect on osteoblast differentiation. The nuclear form of FGF2 functions in mRNA export.

Vascular Epidermal Growth Factor (VEGF):
Vascular endothelial growth factor, originally known as vascular permeability factor (VPF), is a signal protein produced by many cells that stimulates the formation of blood vessels. To be specific, VEGF is a sub-family of growth factors, the platelet-derived growth factor family of cystine-knot growth factors. They are important signaling proteins involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature).

It is part of the system that restores the oxygen supply to tissues when blood circulation is inadequate such as in hypoxic conditions. Serum concentration of VEGF is high in bronchial asthma and diabetes mellitus. VEGF's normal function is to create new blood vessels during embryonic development, new blood vessels after injury, muscle following exercise, and new vessels (collateral circulation) to bypass blocked vessels. It can contribute to disease. Solid cancers cannot grow beyond a limited size without an adequate blood supply; cancers that can express VEGF are able to grow and metastasize. Overexpression of VEGF can cause vascular disease in the retina of the eye and other parts of the body. Drugs such as aflibercept, bevacizumab, ranibizumab, and pegaptanib can inhibit VEGF and control or slow those diseases.

All members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation, although to different sites, times, and extents. The VEGF receptors have an extracellular portion consisting of 7 immunoglobulin-like domains, a single transmembrane spanning region, and an intracellular portion containing a split tyrosine-kinase domain. VEGF-A binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF. The function of VEGFR-1 is less well-defined, although it is thought to modulate VEGFR-2 signaling. Another function of VEGFR-1 may be to act as a dummy/decoy receptor, sequestering VEGF from VEGFR-2 binding (this appears to be particularly important during vasculogenesis in the embryo). VEGF-C and VEGF-D, but not VEGF-A, are ligands for a third receptor (VEGFR-3/Flt4), which mediates lymphangiogenesis. The receptor (VEGFR3) is the site of binding of main ligands (VEGFC and VEGFD), which mediates perpetual action and function of ligands on target cells. Vascular endothelial growth factor-C can stimulate lymphangiogenesis (via VEGFR3) and angiogenesis via VEGFR2. Vascular endothelial growth factor-R3 has been detected in lymphatic endothelial cells in CL of many species, cattle, buffalo and primate.

In addition to binding to VEGFRs, VEGF binds to receptor complexes consisting of both neuropilins and VEGFRs. This receptor complex has increased VEGF signalling activity in endothelial cells (blood vessels). Neuropilins (NRP) are pleiotropic receptors and therefore other molecules may interfere with the signalling of the NRP/VEGFR receptor complexes. For example, Class 3 semaphorins compete with VEGF165 for NRP binding and could therefore regulate VEGF-mediated angiogenesis.

Epidermal growth factor:
Epidermal growth factor (EGF) is a protein that stimulates cell growth and differentiation by binding to its receptor, EGFR. Human EGF is 6-kDa and has 53 amino acid residues and three intramolecular disulfide bonds.

EGF was originally described as a secreted peptide found in the submaxillary glands of mice and in human urine. EGF has since been found in many human tissues, including platelets, submandibular gland (submaxillary gland), and parotid gland. Initially, human EGF was known as urogastrone.

EGF, via binding to its cognate receptor, results in cellular proliferation, differentiation, and survival.

Salivary EGF, which seems to be regulated by dietary inorganic iodine, also plays an important physiological role in the maintenance of oro-esophageal and gastric tissue integrity. The biological effects of salivary EGF include healing of oral and gastroesophageal ulcers, inhibition of gastric acid secretion, stimulation of DNA synthesis as well as mucosal protection from intraluminal injurious factors such as gastric acid, bile acids, pepsin, and trypsin and to physical, chemical and bacterial agents.

EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface. This stimulates ligand-induced dimerization, activating the intrinsic protein-tyrosine kinase activity of the receptor. The tyrosine kinase activity, in turn, initiates a signal transduction cascade that results in a variety of biochemical changes within the cell – a rise in intracellular calcium levels, increased glycolysis and protein synthesis, and increases in the expression of certain genes including the gene for EGFR – that ultimately lead to DNA synthesis and cell proliferation.

Platelet-derived growth factor:
Platelet-derived growth factor (PDGF) is one among numerous growth factors that regulate cell growth and division. In particular, PDGF plays a significant role in blood vessel formation, the growth of blood vessels from already-existing blood vessel tissue, mitogenesis, i.e. proliferation, of mesenchymal cells such as fibroblasts, osteoblasts, tenocytes, vascular smooth muscle cells and mesenchymal stem cells as well as chemotaxis, the directed migration, of mesenchymal cells. Platelet-derived growth factor is a dimeric glycoprotein that can be composed of two A subunits (PDGF-AA), two B subunits (PDGF-BB), or one of each (PDGF-AB).

PDGF is a potent mitogen for cells of mesenchymal origin, including fibroblasts, smooth muscle cells and glial cells. In both mouse and human, the PDGF signalling network consists of five ligands, PDGF-AA through -DD (including -AB), and two receptors, PDGFR-alpha and PDGFR-beta. All PDGFs function as secreted, disulphide-linked homodimers, but only PDGFA and B can form functional heterodimers.

Though PDGF is synthesized, stored (in the alpha granules of platelets), and released by platelets upon activation, it is also produced by other cells including smooth muscle cells, activated macrophages, and endothelial cells.

Recombinant PDGF is used in medicine to help heal chronic ulcers, to heal ocular surface diseases and in orthopedic surgery and periodontics as an alternative to bone autograft to stimulate bone regeneration and repair.

PDGFs are mitogenic during early developmental stages, driving the proliferation of undifferentiated mesenchyme and some progenitor populations. During later maturation stages, PDGF signalling has been implicated in tissue remodelling and cellular differentiation, and in inductive events involved in patterning and morphogenesis. In addition to driving mesenchymal proliferation, PDGFs have been shown to direct the migration, differentiation and function of a variety of specialised mesenchymal and migratory cell types, both during development and in the adult animal. Other growth factors in this family include vascular endothelial growth factors B and C (VEGF-B, VEGF-C) which are active in angiogenesis and endothelial cell growth, and placenta growth factor (PlGF) which is also active in angiogenesis.

PDGF plays a role in embryonic development, cell proliferation, cell migration, and angiogenesis. Over-expression of PDGF has been linked to several diseases such as atherosclerosis, fibrotic disorders and malignancies. Synthesis occurs due to external stimuli such as thrombin, low oxygen tension, or other cytokines and growth factors.

PDGF is a required element in cellular division for fibroblasts, a type of connective tissue cell that is especially prevalent in wound healing. In essence, the PDGFs allow a cell to skip the G1 checkpoints in order to divide. It has been shown that in monocytes-macrophages and fibroblasts, exogenously administered PDGF stimulates chemotaxis, proliferation, and gene expression and significantly augmented the influx of inflammatory cells and fibroblasts, accelerating extracellular matrix and collagen formation and thus reducing the time for the healing process to occur.

In terms of osteogenic differentiation of mesenchymal stem cells, comparing PDGF to epidermal growth factor (EGF), which is also implicated in stimulating cell growth, proliferation, and differentiation, MSCs were shown to have stronger osteogenic differentiation into bone-forming cells when stimulated by epidermal growth factor (EGF) versus PDGF. However, comparing the signalling pathways between them reveals that the PI3K pathway is exclusively activated by PDGF, with EGF having no effect. Chemically inhibiting the PI3K pathway in PDGF-stimulated cells negates the differential effect between the two growth factors, and actually gives PDGF an edge in osteogenic differentiation. Wortmannin is a PI3K-specific inhibitor, and treatment of cells with Wortmannin in combination with PDGF resulted in enhanced osteoblast differentiation compared to just PDGF alone, as well as compared to EGF. These results indicate that the addition of Wortmannin can significantly increase the response of cells into an osteogenic lineage in the presence of PDGF, and thus might reduce the need for higher concentrations of PDGF or other growth factors, making PDGF a more viable growth factor for osteogenic differentiation than other, more expensive growth factors currently used in the field such as BMP2.

PDGF is also known to maintain proliferation of oligodendrocyte progenitor cells (OPCs). It has also been shown that fibroblast growth factor (FGF) activates a signalling pathway that positively regulates the PDGF receptors in OPCs.

Transforming growth factor Beta:
Transforming growth factor beta (TGF-ß) is a multifunctional cytokine belonging to the transforming growth factor superfamily that includes three different mammalian isoforms (TGF-ß 1 to 3, HGNC symbols TGFB1, TGFB2, and TGFB3) and many other signalling proteins. TGFB proteins are produced by all white blood cell lineages.
Activated TGF-ß complexes with other factors to form a serine/threonine kinase complex that binds to TGF-ß receptors. TGF-ß receptors are composed of both type 1 and type 2 receptor subunits. After the binding of TGF-ß, the type 2 receptor kinase phosphorylates and activates the type 1 receptor kinase that activates a signalling cascade. This leads to the activation of different downstream substrates and regulatory proteins, inducing transcription of different target genes that function in differentiation, chemotaxis, proliferation, and activation of many immune cells.

TGF-ß is secreted by many cell types, including macrophages, in a latent form in which it is complexed with two other polypeptides, latent TGF-beta binding protein (LTBP) and latency-associated peptide (LAP). Serum proteinases such as plasmin catalyze the release of active TGF-ß from the complex. This often occurs on the surface of macrophages where the latent TGF-ß complex is bound to CD36 via its ligand, thrombospondin-1 (TSP-1). Inflammatory stimuli that activate macrophages enhance the release of active TGF-ß by promoting the activation of plasmin. Macrophages can also endocytose IgG-bound latent TGF-ß complexes that are secreted by plasma cells and then release active TGF-ß into the extracellular fluid. Among its key functions is regulation of inflammatory processes, particularly in the gut. TGF-ß also plays a crucial role in stem cell differentiation as well as T-cell regulation and differentiation.

Because of its role in immune and stem cell regulation and differentiation, it is a highly researched cytokine in the fields of cancer, auto-immune diseases, and infectious disease. The TGF-ß superfamily includes endogenous growth inhibiting proteins; an increase in expression of TGF-ß often correlates with the malignancy of many cancers and a defect in the cellular growth inhibition response to TGF-ß. Its immunosuppressive functions then come to dominate, contributing to oncogenesis. The dysregulation of its immunosuppressive functions is also implicated in the pathogenesis of autoimmune diseases, although their effect is mediated by the environment of other cytokines present.

Transferrin:
Transferrins are glycoproteins found in vertebrates which bind and consequently mediate the transport of iron (Fe) through blood plasma. They are produced in the liver and contain binding sites for two Fe3+ ions. Human transferrin is encoded by the TF gene and produced as a 76 kDa glycoprotein.

Transferrin glycoproteins bind iron tightly, but reversibly. Although iron bound to transferrin is less than 0.1% (4 mg) of total body iron, it forms the most vital iron pool with the highest rate of turnover (25 mg/24 h). Transferrin has a molecular weight of around 80 kDa and contains two specific high-affinity Fe (III) binding sites. The affinity of transferrin for Fe (III) is extremely high (association constant is 1020 M-1 at pH 7.4) but decreases progressively with decreasing pH below neutrality.
Transferrins are not limited to only binding to iron but also to different metal ions. These glycoproteins are located in various bodily fluids of vertebrates. Some invertebrates have proteins that act like transferrin found in the hemolymph. When not bound to iron, transferrin is known as "apotransferrin".
Transferrins are glycoproteins that are often found in biological fluids of vertebrates. When a transferrin protein loaded with iron encounters a transferrin receptor on the surface of a cell, e.g., erythroid precursors in the bone marrow, it binds to it and is transported into the cell in a vesicle by receptor-mediated endocytosis. The pH of the vesicle is reduced by hydrogen ion pumps (H+ATPases) to about 5.5, causing transferrin to release its iron ions. Iron release rate is dependent on several factors including pH levels, interactions between lobes, temperature, salt, and chelator. The receptor with its ligand bound transferrin is then transported through the endocytic cycle back to the cell surface, ready for another round of iron uptake. Each transferrin molecule has the ability to carry two iron ions in the ferric form (Fe3+).
Transferrin is also associated with the innate immune system. It is found in the mucosa and binds iron, thus creating an environment low in free iron that impedes bacterial survival in a process called iron withholding. The level of transferrin decreases in inflammation.
Albumin:
Albumin is a family of globular proteins, the most common of which are the serum albumins. All of the proteins of the albumin family are water-soluble, moderately soluble in concentrated salt solutions, and experience heat denaturation. Albumins are commonly found in blood plasma and differ from other blood proteins in that they are not glycosylated. Substances containing albumins are called albuminoids.
A number of blood transport proteins are evolutionarily related in the albumin family, including serum albumin, alpha-fetoprotein, vitamin D-binding protein and afamin. This family is only found in vertebrates.
Albumins in general are transport proteins that bind to various ligands and carry them around. Human types include: Human serum albumin is the main protein of human blood plasma. It makes up around 50% of human plasma proteins. It binds water, cations (such as Ca2+, Na+ and K+), fatty acids, hormones, bilirubin, thyroxine (T4) and pharmaceuticals (including barbiturates). Its main function is to regulate the oncotic pressure of blood. The isoelectric point of albumin is 4.7. Alpha-fetoprotein is a fetal plasma protein that binds various cations, fatty acids and bilirubin.
Vitamin D-binding protein binds to vitamin D and its metabolites, as well as to fatty acids. Extracellular matrix protein 1 is a less canonical albumin. It regulates bone mineralization.
Insulin:
Insulin is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the insulin (INS) gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism of carbohydrates, fats and protein by promoting the absorption of glucose from the blood into liver, fat and skeletal muscle cells. In these tissues the absorbed glucose is converted into either glycogen via glycogenesis or fats (triglycerides) via lipogenesis, or, in the case of the liver, into both. Glucose production and secretion by the liver is strongly inhibited by high concentrations of insulin in the blood. Circulating insulin also affects the synthesis of proteins in a wide variety of tissues. It is therefore an anabolic hormone, promoting the conversion of small molecules in the blood into large molecules inside the cells. Low insulin levels in the blood have the opposite effect by promoting widespread catabolism, especially of reserve body fat.

Selenium:
Selenium is a chemical element; it has the symbol Se and atomic number 34. It is a nonmetal (more rarely considered a metalloid) with properties that are intermediate between the elements above and below in the periodic table, sulfur and tellurium, and also has similarities to arsenic. It seldom occurs in its elemental state or as pure ore compounds in Earth's crust.
Selenium is found in metal sulfide ores, where it partially replaces the sulfur. Commercially, selenium is produced as a byproduct in the refining of these ores, most often during production. Minerals that are pure selenide or selenate compounds are known but rare. The chief commercial uses for selenium today are glassmaking and pigments. Selenium is a semiconductor and is used in photocells. Applications in electronics, once important, have been mostly replaced with silicon semiconductor devices. Selenium is still used in a few types of DC power surge protectors and one type of fluorescent quantum dot.
Although trace amounts of selenium are necessary for cellular function in many animals, including humans, both elemental selenium and (especially) selenium salts are toxic in even small doses, causing selenosis. Selenium is listed as an ingredient in many multivitamins and other dietary supplements, as well as in infant formula, and is a component of the antioxidant enzymes glutathione peroxidase and thioredoxin reductase (which indirectly reduce certain oxidized molecules in animals and some plants) as well as in three deiodinase enzymes. Selenium requirements in plants differ by species, with some plants requiring relatively large amounts and others apparently not requiring any.
Selenium may be measured in blood, plasma, serum, or urine to monitor excessive environmental or occupational exposure, to confirm a diagnosis of poisoning in hospitalized victims, or investigate a suspected case of fatal overdose. Some analytical techniques are capable of distinguishing organic from inorganic forms of the element. Both organic and inorganic forms of selenium are largely converted to monosaccharide conjugates in the body prior to elimination in the urine. Cancer patients receiving daily oral doses of selenothionine may achieve very high plasma and urine selenium concentrations.

Fetuin
Fetuins are blood proteins that are made in the liver and secreted into the bloodstream. They belong to a large group of binding proteins mediating the transport and availability of a wide variety of cargo substances in the bloodstream. Fetuin-A is a major carrier protein of free fatty acids in the circulation. The best known representative of carrier proteins is serum albumin, the most abundant protein in the blood plasma of adult animals. Fetuin is more abundant in fetal blood, hence the name "fetuin". Fetal bovine serum contains more fetuin than albumin, while adult serum contains more albumin than fetuin.

Vitronectin
Vitronectin (VTN or VN) is a glycoprotein of the hemopexin family which is synthesized and excreted by the liver, and abundantly found in serum, the extracellular matrix and bone. In humans it is encoded by the VTN gene.
Vitronectin binds to integrin alpha-V beta-3 and thus promotes cell adhesion and spreading. It also inhibits the membrane-damaging effect of the terminal cytolytic complement pathway and binds to several serpins (serine protease inhibitors). It is a secreted protein and exists in either a single chain form or a clipped, two chain form held together by a disulfide bond. Vitronectin has been speculated to be involved in hemostasis and tumor malignancy.
The somatomedin B domain of vitronectin binds to plasminogen activator inhibitor-1 (PAI-1), and stabilizes it. Thus vitronectin serves to regulate proteolysis initiated by plasminogen activation. In addition, vitronectin is a component of platelets and is, thus, involved in hemostasis. Vitronectin contains an RGD (45-47) sequence, which is a binding site for membrane-bound integrins, e.g., the vitronectin receptor, which serve to anchor cells to the extracellular matrix. The Somatomedin B domain interacts with the urokinase receptor, and this interaction has been implicated in cell migration and signal transduction. High plasma levels of both PAI-1 and the urokinase receptor have been shown to correlate with a negative prognosis for cancer patients. Cell adhesion and migration are directly involved in cancer metastasis, which provides a probable mechanistic explanation for this observation.

Fibronectin
Fibronectin is a high-molecular weight (~500-~600 kDa) glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. Fibronectin also binds to other extracellular matrix proteins such as collagen, fibrin, and heparan sulfate proteoglycans.
Fibronectin exists as a protein dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds. The fibronectin protein is produced from a single gene, but alternative splicing of its pre-mRNA leads to the creation of several isoforms.
Fibronectin plays a major role in cell adhesion, growth, migration, and differentiation, and it is important for processes such as wound healing and embryonic development. Altered fibronectin expression, degradation, and organization has been associated with a number of pathologies, including cancer, arthritis, and fibrosis.
Fibronectin has numerous functions that ensure the normal functioning of vertebrate organisms. It is involved in cell adhesion, growth, migration, and differentiation. Cellular fibronectin is assembled into the extracellular matrix, an insoluble network that separates and supports the organs and tissues of an organism.
Fibronectin plays a crucial role in wound healing. Along with fibrin, plasma fibronectin is deposited at the site of injury, forming a blood clot that stops bleeding and protects the underlying tissue. As repair of the injured tissue continues, fibroblasts and macrophages begin to remodel the area, degrading the proteins that form the provisional blood clot matrix and replacing them with a matrix that more resembles the normal, surrounding tissue. Fibroblasts secrete proteases, including matrix metallo-proteinases, that digest the plasma fibronectin, and then the fibroblasts secrete cellular fibronectin and assemble it into an insoluble matrix. Fragmentation of fibronectin by proteases has been suggested to promote wound contraction, a critical step in wound healing. Fragmenting fibronectin further exposes its V-region, which contains the site for a4ß1 integrin binding. These fragments of fibronectin are believed to enhance the binding of a4ß1 integrin-expressing cells, allowing them to adhere to and forcefully contract the surrounding matrix.
Fibronectin is necessary for embryogenesis, and inactivating the gene for fibronectin results in early embryonic lethality. Fibronectin is important for guiding cell attachment and migration during embryonic development. In mammalian development, the absence of fibronectin leads to defects in mesodermal, neural tube, and vascular development. Similarly, the absence of a normal fibronectin matrix in developing amphibians causes defects in mesodermal patterning and inhibits gastrulation.
Fibronectin is also found in normal human saliva, which helps prevent colonization of the oral cavity and pharynx by pathogenic bacteria.

GLUT1:
Glucose transporter 1 (or GLUT1), also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1), is a uniporter protein that in humans is encoded by the SLC2A1 gene. GLUT1 facilitates the transport of glucose across the plasma membranes of mammalian cells. This gene encodes a facilitative glucose transporter that is highly expressed in erythrocytes and endothelial cells, including cells of the blood–brain barrier. The encoded protein is found primarily in the cell membrane and on the cell surface, where it can also function as a receptor for human T-cell leukemia virus (HTLV) I and II. GLUT1 accounts for 2 percent of the protein in the plasma membrane of erythrocytes.

Mutations in this gene can cause GLUT1 deficiency syndrome 1, GLUT1 deficiency syndrome 2, idiopathic generalized epilepsy 12, dystonia 9, and stomatin-deficient cryohydrocytosis.

The SLC2A1 gene is located on the p arm of chromosome 1 in position 34.2 and has 10 exons spanning 33,802 base pairs. The gene produces a 54.1 kDa protein composed of 492 amino acids. It is a multi-pass protein located in the cell membrane. This protein lacks a signal sequence; its C-terminus, N-terminus, and the very hydrophilic domain in the protein's center are all predicted to lie on the cytoplasmic side of the cell membrane.

GLUT1 behaves as a Michaelis–Menten enzyme and contains 12 membrane-spanning alpha helices, each containing 20 amino acid residues. A helical wheel analysis shows that the membrane-spanning alpha-helices are amphipathic, with one side being polar and the other side hydrophobic. Six of these membrane-spanning helices are believed to bind together in the membrane to create a polar channel in the center through which glucose can traverse, with the hydrophobic regions on the outside of the channel adjacent to the fatty acid tails of the membrane.

Energy-yielding metabolism in erythrocytes depends on a constant supply of glucose from the blood plasma, where the glucose concentration is maintained at about 5mM. Glucose enters the erythrocyte by facilitated diffusion via a specific glucose transporter, at a rate of about 50,000 times greater than uncatalyzed transmembrane diffusion. The glucose transporter of erythrocytes (called GLUT1 to distinguish it from related glucose transporters in other tissues) is a type III integral protein with 12 hydrophobic segments, each of which is believed to form a membrane-spanning helix. The detailed structure of GLUT1 is not known yet, but one plausible model suggests that the side-by-side assembly of several helices produces a transmembrane channel lined with hydrophilic residues that can hydrogen-bond with glucose as it moves through the channel.

GLUT1 is responsible for the low level of basal glucose uptake required to sustain respiration in all cells. Expression levels of GLUT1 in cell membranes are increased by reduced glucose levels and decreased by increased glucose levels.

GLUT1 is also a major receptor for uptake of Vitamin C as well as glucose, especially in non-vitamin C producing mammals as part of an adaptation to compensate by participating in a Vitamin C recycling process. In mammals that do produce Vitamin C, GLUT4 is often expressed instead of GLUT1.

Sodium-glucose linked transporter (SGLT):
Sodium-dependent glucose cotransporters (or sodium-glucose linked transporter, SGLT) are a family of glucose transporter found in the intestinal mucosa (enterocytes) of the small intestine (SGLT1) and the proximal tubule of the nephron (SGLT2 in PCT and SGLT1 in PST). They contribute to renal glucose reabsorption. In the kidneys, 100% of the filtered glucose in the glomerulus has to be reabsorbed along the nephron (98% in PCT, via SGLT2). If the plasma glucose concentration is too high (hyperglycemia), glucose passes into the urine (glucosuria) because SGLT are saturated with the filtered glucose.

The sodium-glucose linked transporters (SGLTs) are responsible for the active transport of glucose across cell membranes. SGLT1 and SGLT2 are the well-studied members of this family. Both SGLT1 and SGLT2 function as symporters, utilizing the energy from the sodium gradient created by the Na+/K+ ATPase to transport glucose against its concentration gradient.

The transport of glucose across the proximal tubule cell membrane involves a complex process of secondary active transport (also known as co-transport). This process begins with the Na+/K+ ATPase on the basolateral membrane. This enzyme uses ATP to pump 3 sodium ions out of the cell into the blood while bringing 2 potassium ions into the cell. This action creates a sodium concentration gradient across the cell membrane, with a lower concentration inside the cell compared to both the blood and the tubular lumen.

SGLT proteins utilize this sodium gradient to transport glucose across the apical membrane into the cell, even against the glucose concentration gradient. This mechanism is an example of secondary active transport. Once inside the cell, glucose is then moved across the basolateral membrane into the peritubular capillaries by members of the GLUT family of glucose uniporters.

SGLT1 and SGLT2 are classified as symporters because they move sodium and glucose in the same direction across the membrane. To maintain this process, the Sodium–hydrogen antiporter plays a crucial role in replenishing intracellular sodium levels. Consequently, the net effect of glucose transport is coupled with the extrusion of protons from the cell, with sodium serving as an intermediate in this process.

While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein. The invention should therefore not be limited by the above described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention. The invention shall now be described with reference to the following specific examples. It should be noted that the example(s) appended below illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the present invention.
These and other aspects of the invention may become more apparent from the examples set forth herein below. These examples are provided merely as illustrations of the invention and are not intended to be construed as a limitation thereof.
EXAMPLES

EXAMPLE 1: Preparation of a Biochemical composition (Form A) as a FBS alternative

A cell culture formulation is prepared to overcome the limitations of FBS. An alternative to FBS is prepared from animal-origin free source. Such an alternative is designed to emulate the beneficial properties of FBS while mitigating the associated risks. Cell culture media Formulation A (Form A) is prepared at pH 7.3 to optimize cell proliferation and viability which is ideal for general cell culture and high-yield bioproduction. Form A is specifically optimized for the proliferation of HEK293 and HaCaT cell lines. The composition of Form A comprises:

Component Compound Type Concentration
DMEM Base medium 450 ml
F12 Base medium 450 ml
FGF2 Growth Factor 10 ng/ml
EGF Growth Factor 5 ng/ml
VEGF Growth Factor 5 ng/ml
Recombinant Human Albumin Carrier Protein 1% w/v
Synthetic Phospholipid Lipid 30 ug/ml
Iron Trace element 2 uM/ml
Zinc Trace element 2 uM/ml
Selenium Trace element 2 uM/ml
HEPES Buffering Agent 10 mM/ml
Sodium Bicarbonate Buffering Agent 10 mM/ml
Double Distilled Water Diluent/solvent 100 ml

EXAMPLE 2: Preparation of a Biochemical composition (Form B) as a FBS alternative

A cell culture formulation is prepared to overcome the limitations of FBS. An alternative to FBS is prepared from animal-origin free source. Such an alternative is designed to emulate the beneficial properties of FBS while mitigating the associated risks. Cell culture media Formulation B (Form B) is prepared to optimize and regulate cell differentiation. The Form B uses cholesterol supplements to enhance membrane fluidity which is crucial for the dynamic structural changes that occur during cell differentiation. Form B is specifically optimized for the differentiation of C2C12 cell line into myotubes (precursor cell for forming the skeletal muscles). The composition of Form B comprises:

Component Compound Type Concentration
DMEM Base medium 450 ml
High glucose solution Base medium 450 ml
TGF-ß1 Growth Factor 2 ng/ml
PDGF Growth Factor 10 ng/ml
Bovine Serum Albumin Carrier Protein 0.5% w/v
Cholesterol Supplement Lipid 10 ug/ml
Copper Trace element 1 uM/ml
Sodium Bicarbonate Buffering Agent 12 mM/ml
Double Distilled Water Diluent/solvent 100 ml

EXAMPLE 3: Preparation of a Biochemical composition (Form C) as a FBS alternative

A cell culture formulation is prepared to overcome the limitations of FBS. An alternative to FBS is prepared from animal-origin free source. Such an alternative is designed to emulate the beneficial properties of FBS while mitigating the associated risks. Cell culture media Formulation C (Form C) at pH 7.4 is prepared to demonstrate 10% increase in efficiency compared to traditional FBS across all tested cell lines. This ultimately offers a significant advantage in therapeutic cell production and High-stakes cell line research. The composition of Form C comprises:

Component Compound Type Concentration
DMEM Base medium 450 ml
F12 Base medium 450 ml
FGF2 Growth Factor 15 ng/ml
EGF Growth Factor 10 ng/ml
PDGF Growth Factor 15 ng/ml
Recombinant Human Albumin Carrier Protein 1% w/v
Advanced Lipid Complex Lipid 50 ug/ml
Manganese Trace element 3 uM/ml
HEPES Buffering Agent 15 mM/ml
Sodium Bicarbonate Buffering Agent 15 mM/ml
Double Distilled Water Diluent/solvent 100 ml

EXAMPLE 4: Cell Proliferation Assay
The current experiment is performed to establish that the current patent has the ability to support a robust cell growth compared to conventional FBS-based media. The serum-free medium as disclosed in the current invention is tested against conventional FBS-supplemented media (Positive Control) and base medium without supplements (Negative Control). Human embryonic kidney cells (HEK293), Chinese hamster ovary cells (CHO-K1) and Human mesenchymal stem cells (hMSCs) cell lines were grown at a seed cell density of 1 x 104 cells per well in a 96-well plate in each of the aforementioned mediums. Cells were incubated in the respective media under standard conditions at 37 °C, 5% CO2. Subsequently, MTT assay was performed on each cell to analyze mitochondrial activity to determine the cell viability and proliferation. Formazan crystals were added to each of the wells to analyze mitochondrial activity and cell viability of the cell lines. Resultantly, the serum-free medium showed higher cell viability and proliferation with respect to conventional FBS-supplemented medium. While the negative control showed minimal proliferation. This was analyzed by measuring absorbance at 570 nm using a microplate reader.

EXAMPLE 5: Cell Doubling Time
This protocol outlines a systematic approach for evaluating the cumulative doubling times of HEK293, HaCaT, and C2C12 cell lines cultured in four distinct media formulations: FBS, Form A, Form B, and Form C. Over a 7-day period, cells were seeded at a known initial density in T-25 flasks and maintained under standard culture conditions, with daily monitoring and media changes every 48 hours or as needed. Cells were counted at regular 24-hour intervals using a hemocytometer or automated cell counter, and viability was confirmed via trypan blue exclusion. Doubling times were calculated using a logarithmic formula based on the initial and final cell counts. Growth curves were plotted, and proliferation rates were compared across media types. Throughout the experiment, rigorous aseptic techniques were observed, and comprehensive documentation was maintained to ensure data integrity and regulatory compliance.

Day Condition Doubling Time (hrs) CPD (Cumulative Population Doubling)
HEK293 Cell line
1 FBS 24 1
1 Form A 26 0.92
1 Form B 30 0.8
1 Form C 22 1.09
2 FBS 24 2
2 Form A 26 1.85
2 Form B 30 1.6
2 Form C 22 2.18
3 FBS 24 3
3 Form A 26 2.77
3 Form B 30 2.4
3 Form C 22 3.27
4 FBS 24 4
4 Form A 26 3.69
4 Form B 30 3.2
4 Form C 22 4.36
5 FBS 24 5
5 Form A 26 4.62
5 Form B 30 4
5 Form C 22 5.45
6 FBS 24 6
6 Form A 26 5.54
6 Form B 30 4.8
6 Form C 22 6.55
7 FBS 24 7
7 Form A 26 6.46
7 Form B 30 5.6
7 Form C 22 7.64

Day Condition Doubling Time (hrs) CPD (Cumulative Population Doubling)
C2C12 Cell line
1 FBS 26 0.92
1 Form A 28 0.86
1 Form B 32 0.75
1 Form C 24 1
2 FBS 26 1.85
2 Form A 28 1.71
2 Form B 32 1.5
2 Form C 24 2
3 FBS 26 2.77
3 Form A 28 2.57
3 Form B 32 2.25
3 Form C 24 3
4 FBS 26 3.69
4 Form A 28 3.43
4 Form B 32 3
4 Form C 24 4
5 FBS 26 4.62
5 Form A 28 4.29
5 Form B 32 3.75
5 Form C 24 5
6 FBS 26 5.54
6 Form A 28 5.14
6 Form B 32 4.5
6 Form C 24 6
7 FBS 26 6.46
7 Form A 28 6
7 Form B 32 5.25
7 Form C 24 7

Day Condition Doubling Time (hrs) CPD (Cumulative Population Doubling)
HACAT Cell line
1 FBS 30 0.8
1 Form A 32 0.75
1 Form B 36 0.67
1 Form C 28 0.86
2 FBS 30 1.6
2 Form A 32 1.5
2 Form B 36 1.33
2 Form C 28 1.71
3 FBS 30 2.4
3 Form A 32 2.25
3 Form B 36 2
3 Form C 28 2.57
4 FBS 30 3.2
4 Form A 32 3
4 Form B 36 2.67
4 Form C 28 3.43
5 FBS 30 4
5 Form A 32 3.75
5 Form B 36 3.33
5 Form C 28 4.29
6 FBS 30 4.8
6 Form A 32 4.5
6 Form B 36 4
6 Form C 28 5.14
7 FBS 30 5.6
7 Form A 32 5.25
7 Form B 36 4.67
7 Form C 28 6

EXAMPLE 6: Cytokine Production Assay
This experiment was conducted to assess the ability of the serum free medium to support cytokine production, indicating functional cell performance and suitability for therapeutic applications. The animal-serum free medium as disclosed in the current invention is tested against the FBS-supplemented medium with Lipopolysaccharide (LPS) stimulation (Positive Control) and base medium without supplements and no LPS stimulation (Negative Control). Human monocyte-derived macrophages (THP-1) and Peripheral blood mononuclear cells (PBMC) cell lines were grown at a seed cell density of 1 x 106 cells/ml in a 24-well plate in each of the aforementioned medium. The cells were incubated for 24hr under standard conditions at 37 °C and 5% CO2. Consequently, the cytokine production was measured by collecting supernatant after 24hr incubation period. The supernatant analysis by ELISA test was performed for IL-6, TNF-a, and IL-10 which revealed that cytokine levels in the serum-free medium are comparable to those in FBS-supplemented medium. Minimal cytokine production was seen in the negative control.

EXAMPLE 7: Cytotoxicity Assay
This experiment was conducted to determine the safety and biocompatibility of the serum-free medium by measuring its cytotoxic effects on cell lines. HaCaT (human keratinocytes) and hMSCs (human mesenchymal stem cells) were grown at a seed cell density of 5 x 104 cells/well in a 96-well plate in 3 different media namely, Serum-free medium as disclosed in the present invention, FBS-supplemented medium as positive control and Base medium without supplements as the negative control. The cultures were incubated for a total of 48h under standard conditions at 37 °C and 5% CO2. Consequently, supernatant samples were collected at 24h and 48h for analysis of cytotoxicity. Cytotoxicity was assessed by using Lactate dehydrogenase (LDH) release assay. Absorbance was measured at 490 nm to quantify LDH release to assess the cell membrane integrity.

EXAMPLE 8: ED50 Determination Assay
This experiment was conducted to determine the effective dose (ED50) of recombinant growth factors in the serum-free medium to maximize cell viability and proliferation. Human embryonic kidney cells (HEK293) and Chinese hamster ovary cells (CHO-K1) were cultured at seed cell density of 5 x 104 cells/well in a 96-well plate in 3 different media namely Serum-free medium with varying concentrations of individual growth factors, FBS-supplemented medium as positive control and base medium without growth factors as negative control. The cells were incubated for 48hr under standard condition at 37 °C and 5% CO2.The growth factors used in this experiment were FGF2, VEGF, EGF, TGF-ß1 and PDGF. MTT assay was performed to evaluate cell viability at different recombinant growth factor concentrations ranging between 0.1 to 100 ng/ml. Absorbance was measured at 570 nm and a plot dose-response curves were prepared to determine ED50 values for each growth factor.
,CLAIMS:We Claim,

1. A serum-free cell culture media for proliferation, differentiation and to support multiple mammalian cell lines comprising base medium as carbon source, carrier proteins for uptake of nutrients by mammalian cells, recombinant cell growth factors to optimize cell proliferation, buffering agents to maintain pH, synthetic lipids for providing energy and help in cell membrane formation, trace elements and supplementary additives as nutrient source, wherein:
a. a base medium suitable for mammalian cell culture is selected from Minimal Essential Medium (MEM), F12 nutrient mixture and High Glucose support medium or combination thereof;
b. a carrier protein selected from a class of Recombinant proteins, Glucose transporter proteins, Iron transporter protein and Glycoproteins;
c. a combination of recombinant cell growth factors selected from the family of Insulin-like Growth Factors and Epidermal Growth Factors;
d. a combination of synthetic buffering agents;
e. a synthetic lipid source selected from the class of simple, complex and miscellaneous lipids;
f. a combination of trace elements selected from the class of essential and non-essential trace elements; and
g. supplementary additives with plant peptide extracts.

2. The serum-free cell culture medium as claimed in claim 1, wherein the base medium is selected from Minimal Essential Medium (MEM), F12 nutrient medium, High Glucose Support medium in a ratio of 1:1.

3. The serum-free cell culture medium as claimed in claim 1, wherein the recombinant cell growth factors selected from the Epidermal Growth Factor family are Fibroblast growth factor 2 (FGF-2) and Epidermal growth factor (EGF) or combination thereof in an amount of 0.1 to 100 ng/ml.

4. The serum-free cell culture medium as claimed in claim 1, wherein the recombinant cell growth factors selected from the Insulin-like Growth Factor family are Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor ß1 (TGF-ß1), Platelet-derived Growth Factor (PDGF) or combination thereof in an amount of 0.1 to 100 ng/ml.

5. The serum-free cell culture medium as claimed in claim 1, wherein the carrier protein from the class of Recombinant Protein is preferably selected from animal-free sources such as recombinant human albumin, recombinant Bovine serum albumin, or combination thereof present in an amount of 0.1% to 12.0% w/v.

6. The serum-free cell culture medium as claimed in claim 1, wherein the carrier protein from the class of Glucose transport protein is selected from GLUT1, Sodium-dependent Glucose transporters (SGLTs) or combination thereof present in an amount of 0.1% to 12.0% w/v.

7. The serum-free cell culture medium as claimed in claim 1, wherein the carrier protein from the class of Iron transporters is selected from transferrins, lactoferrin, ferroportin or combination thereof present in an amount of 0.1% to 12.0% w/v.

8. The serum-free cell culture medium as claimed in claim 1, wherein the carrier protein from the class of Glycoproteins is selected from Fetuin, Vitronectin or combination thereof present in an amount of 0.1% to 12.0% w/v.

9. The serum-free cell culture medium as claimed in claims 1, wherein the synthetic lipid source of simple lipids is selected from triglycerides and waxes or combination thereof present in an amount of 10 to 50 µg/ml.

10. The serum-free cell culture medium as claimed in claim 1, wherein the synthetic lipid source of complex lipids is selected from phospholipids, glycolipids and lipoproteins or combination thereof present in an amount of 10 to 50 µg/ml.

11. The serum-free cell culture medium as claimed in claim 1, wherein the synthetic lipid source of miscellaneous lipids is selected from Recombinant albumin, liposomes, carotenoids, fat-soluble vitamins or combination thereof present in an amount of 10 to 50 µg/ml.

12. The serum-free cell culture medium as claimed in claims 1, wherein the essential trace elements are selected from Iron, selenium, zinc, copper, manganese or combination thereof present in an amount of 1 to 5 µM.

13. The serum-free cell culture medium as claimed in claims 1, wherein the non-essential trace elements are selected from aluminum, nickel, bromine, silver or combination thereof.

14. The serum-free cell culture medium as claimed in claims 1, wherein the synthetic buffering agent is selected from HEPES, MOPS, Sodium bicarbonate, TAPS, phosphate buffer solution (PBS) or combination thereof present in an amount of 10 to 25 mM.

15. The serum-free cell culture medium as claimed in claims 1, wherein the supplementary additives are selected from amino acids, vitamins, antioxidants, Plant peptide extracts or combination thereof in an amount 0.1% to 12.0% w/v.

16. The supplementary additives mixed with the serum-free cell culture medium as claimed in claim 14, wherein the essential amino acids are selected from isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, or combination thereof.

17. The supplementary additives mixed with the serum-free cell culture medium as claimed in claim 14, wherein the non-essential amino acids are selected from glycine, alanine, proline or combination thereof.

18. The supplementary additives mixed with the serum-free cell culture medium as claimed in claim 14, wherein the vitamins are selected Vitamin B (biotin, folic acid, B12, riboflavin, thiamine), Vitamin C, or other types of vitamins added in accordance with cell lines grown.

19. The supplementary additives mixed with the serum-free cell culture medium as claimed in claim 14, wherein the antioxidant is selected from glutathione, Vitamin C, ascorbic acid, phenol red, luteolin, kaempferol, resveratrol or combination thereof.

20. The supplementary additives mixed with the serum-free cell culture medium as claimed in claim 14, wherein plant peptide extract is selected from wheat gluten proteins, wheat a-gliadin hydrolysate, Rape seed hydrolysate, Rice endosperm protein hydrolysate, soy ß-conglycinin, Corn gluten meal hydrolysate, black soybean peptides, Canary seed protein, Mung bean protein hydrolysate, oat bran protein hydrolysate, chickpea albumin hydrolysate, hempseed protein hydrolysate or combination thereof.