DESC:OPTIMIZED HEMATOPOIETIC STEM CELL CFU ASSAY

FIELD OF THE INVENTION

[0001] The invention generally relates to a method of optimizing hematopoietic stem cell CFU assay. More specifically, the invention relates to a method of providing a combination of antibiotics for inhibiting microbial contamination in hematopoietic stem cell CFU assay.

BACKGROUND OF THE INVENTION

[0002] Human hematopoietic stem cells obtained from various sources, including bone marrow, cord blood or peripheral blood typically undergoes a CFC/CFU (colony forming cell/colony forming unit) assay. A CFU assay enables the assessment and study of the proliferation and differentiation abilities of hematopoietic progenitor cells based on ability of the hematopoietic progenitor cells to form colonies in a semi-solid medium.

[0003] A CFU assay procedure involves the plating of a cell suspension in a semi-solid cell culture medium under predefined optimal conditions for identifying and counting discrete colonies containing recognizable progeny. Considering the harvesting and collection of the various sources of bone marrow, cord blood or peripheral blood is done from hospital environments, susceptibility to microbial contamination chances are high.

[0004] Microbial contamination encountered during Hematopoietic stem cell CFU assay is detrimental to the growth and expansion of hematopoietic progenitor cells as the microbial contamination depletes the nutrients from the growth media interfering with the growth and proliferation of the hematopoietic stem cells. Furthermore, microbial contamination causes accumulation of toxic metabolites into the media which leads to change in the pH of the media, making the growth medium highly unfavorable for the hematopoietic stem cells proliferation.

[0005] Furthermore, the colonies in the CFU assay are not able to grow within the stipulated period of 14 days, instead the plates are contaminated due to bacterial intervention. Contaminated plates of the CFU assay with hematopoietic stem cells hampers the selection of promising cord blood units for future storage as well as selection of one or more suitable units for transplantation.

BRIEF DESCRIPTION OF THE FIGURES

[0006] The accompanying figures where like reference numerals refer to identical or functionally similar elements throughout the separate views and which together with the detailed description below are incorporated in and form part of the specification, serve to further illustrate various embodiments and to explain various principles and advantages all in accordance with the invention.

[0007] FIG. 1 represents a flowchart illustrating the procedural steps involved in a hematopoietic stem cell CFU assay.

[0008] FIG. 2 represents microbial contamination in a hematopoietic stem cell CFU cell culture assay.

[0009] Fig. 3A and 3B are representative of hematopoietic stem cell colonies after 7 days and 14 days of culture of umbilical cord blood (CB) mononuclear cells in HSC CFU assay Media supplemented with the synergistic antibiotic composition.

[0010] Figure 4 is representative of CFU-GM and BFU-E colonies in HSC CFU assay Media supplemented with the synergistic antibiotic composition.

[0011] Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help to improve understanding of embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0012] Before describing in detail embodiments that are in accordance with the invention, it should be observed that the embodiments reside primarily in combinations of method steps and composition related to inhibiting microbial contamination in effective proliferation and expansion of hematopoietic stem cell CFU assay.

[0013] In this document, relational terms such as first and second, top and bottom, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article or composition that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article or composition. An element proceeded by “comprises …a” does not, without more constraints, preclude the existence of additional identical elements in the process, method, article or composition that comprises the element.

[0014] Various embodiments of the invention provide a method for inhibiting microbial contamination encountered by human umbilical cord blood cells, thereby optimizing cell culture assay in a Hematopoietic stem cell CFU Assay. Inhibiting the microbial contamination enables the selection of promising cord blood units for future storage as well as selection of one or more suitable units for transplantation despite of microbial contamination.

[0015] The contamination encountered during umbilical cord blood collection are due to various factors, including, but not limited to, vaginal flora, unhygienic labour rooms environment, non-aseptic surgical instruments and lack of appropriate physician skill for cord blood collection procedure. The bacterial microbes include a plurality of gram positive bacteria as well as a plurality of gram negative bacteria.

[0016] Accordingly, a synergistic antibiotic composition comprising a mixture of ß-lactam antibiotic and an Aminoglycoside along with a ß-lactamase inhibitor is supplemented to a CFU assay media to effectively inhibit microbial contamination. The CFU assay media is an optimized semi-solid Methylcellulose-based CFU assay media including, but not limited to, methylcellulose, fetal bovine serum, bovine serum albumin, L-glutamine, a reducing agent such as 2-mercaptoethanol, basal medium such as lscove's MDM and recombinant cytokines. Further, the recombinant cytokines include, but are not limited to, IL-3, IL-6, SCF, GM-CSF, G-CSF and EPO.

[0017] In some embodiments, the synergistic antibiotic composition includes a ß-lactamase inhibitor and a mixture of penicillin and streptomycin. The ß-lactamase inhibitor is selected from a group of sulbactam, clavulanate, tazobactam and avibactam. In a preferred embodiment, the synergistic antibiotic composition includes a mixture of penicillin and streptomycin along with sulbactam, wherein the concentration range of sulbactam supplemented is 35-50 µg/ml and the concentration range of penicillin and streptomycin are 50-200 I.U./mL and 50-200 µg/ml respectively to constitute the penicillin and streptomycin mixture. In a preferred embodiment, the concentration amount of penicillin and streptomycin are 100 I.U./mL and 100 µg/ml respectively.

[0018] Accordingly, ß-lactamase inhibitor acts as an adjuvant to the ß-lactam antibiotics and prevents bacterial degradation of ß-lactam antibiotics. The beta-lactam ring of the ß-lactamase inhibitor irreversibly binds to a particular ß-lactam antibiotic at the active site or near the active site for blocking enzyme activity and preventing metabolism of the ß-lactam antibiotic by a ß-lactamase enzyme produced by resistant bacteria.

[0019] In an embodiment, the mixture of antibiotics may be further selected from a group of penicillin, phenethicillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, ciclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, mezlocillin, mecillinam, andinocillin, cephalosporin and a derivative thereof, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, piromidic acid, ciprofloxacin, cinoxacin, norfloxacin, perfloxacin, rosaxacin, ofloxacin, enoxacin, pipemidic acid, clavulanic acid, ß-bromopenisillanic acid, ß-chloropenisillanic acid, 6-acetylmethylene-penisillanic acid, cephoxazole, sultampicillin, formaldehyde hydrate esters of amdinocillin, aztreonam, sulfazethin, isosulfazethin, norcardicin, m-carboxyphenyl, phenylacetamidophosphonic acid methyl, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline. The ß-lactamase inhibitor may be further selected from a group of clavulanate, avibactam and tazobactam.

[0020] In the preferred embodiment of sulbactam and a mixture of penicillin and streptomycin, the synergistic penicillin-streptomycin antibiotic mixture has a wide range of broad-spectrum bactericidal activity against gram-positive bacteria as well as gram-negative bacteria. The combination of sulbactam and penicillin-streptomycin antibiotic mixture enables sulbactam to function as an adjuvant to effectively target drug resistant microbes resulting from inhibitory activity against plasmid mediated beta-lactamases, thereby addressing transferred drug resistance. The gram-positive bacteria, include, but are not limited to, Enterococcus faecalis, Bacillus sp., Methicillin Resistant Staphylococcus aureus [MRSA] and Methicillin Resistant Coagulase Negative Staphylococcus sp. The gram-negative bacteria, include, but are not limited to, Enterobacter sp., Acinetobacter baumannii, Pseudomonas sp., Escherichia coli, Stenotrophomonas maltophilia, and Klebsiella pneumoniae.

[0021] In yet another embodiment, the synergistic antibiotic composition includes a ß-lactamase inhibitor at a concentration of 50 µg/ml supplemented to the Methylcellulose-based CFU assay media. The supplemented Methylcellulose-based CFU assay media comprising the ß-lactamase inhibitor based synergistic antibiotic composition is effective against a group of gram negative bacteria. The group of gram negative bacteria include, but are not limited to, Escherichia coli, Acinetobacter baumanii and the likes thereof.

[0022] FIG. 1 is representative of a flow chart illustrating the procedural steps involved in collection and processing of cord blood and the procedural steps involved in a hematopoietic stem cell CFU assay.

[0023] As illustrated in FIG. 1, at an initial step 102, umbilical cord blood sample collected from a hospital undergoes processing steps. The umbilical cord blood sample are collected from a hospital using aseptic techniques, wherein sterile gloves are employed by an individual collecting the cord blood, following which the umbilical cord is cleaned using 70% isopropyl alcohol or Povidone-iodine. The umbilical cord blood is collected in the collection bag, wherein the collection bag attached with a needle is inserted into the umbilical cord vein allowing the umbilical cord blood to flow into the collection bag. The influxed blood is gently mixed with an anticoagulant present in the collection bag. In an ensuing step, the collection bag is packed in a controlled temperature kit box and sent to a central processing unit to be further transferred to a biosafety cabinet of the processing area. The central processing unit can be a laboratory or a cord blood processing center. Additionally, the umbilical cord blood sample is subjected to sedimentation for collecting a supernatant leucocyte rich plasma. Further, while processing the umbilical cord blood sample, the supernatant leucocyte rich plasma is centrifuged, and a leukocyte concentrate is obtained. In a concluding step, a part volume of this leucocyte concentrate is used for the CFU assay and the remaining sample is kept for cryopreservation.

[0024] In an embodiment, a cryoprotectant is added for cryopreservation and the resultant sample is stored below -1500C till retrieval.

[0025] At step 104, the collected leukocyte concentrate undergoes a hematopoietic stem cell CFU assay. The collected umbilical cord blood cells may be contaminated thereby contaminating the hematopoietic stem cell CFU assay. In a subsequent step 106, the leukocyte concentrate is counted on an automated cell counter.

[0026] Further referring to Fig. 1, in a preferred embodiment, an optimized CFU-Assay with an antimicrobial cell culture assay is performed, wherein the optimized CFU-Assay with the antimicrobial cell culture assay includes a plurality of steps. The processed cell sample of umbilical cord blood cells are diluted to an appropriate cell concentration in (IMDM+2 % FBS) medium to 2*104 cells at a step 108, making a primary dilution and secondary dilution in the optimized methylcellulose medium. Subsequently, on the night before the testing is to be conducted, the aliquots of methylcellulose medium are thawed at 4º C. The aliquots of methylcellulose medium are homogenized and the homogenized Methylcellulose-based CFU assay media are transferred into 4 ml aliquots in 15 ml round bottom tubes. Further, 35 mm culture dishes are labelled on an incubator tray. In a following step, antibiotic dilutions of the ß-lactamase inhibitor at different concentrations including 35 µg/ml, 40 µg/ml and 50 µg/ml are prepared. Simultaneously, antibiotic dilutions of a mixture of penicillin and streptomycin, the mixture comprising a concentration amount of 100 I.U./ml of penicillin and 100 µg/ml of streptomycin are prepared. A synergistic antibiotic combination is added to the homogenized Methylcellulose-based CFU assay media, at step 110 along with a sample from the secondary dilution. In an ensuing step 112, the diluted cell sample of umbilical cord blood cells are added to pre-aliquoted tubes of Methylcellulose-based CFU assay media with supplemented antibiotic composition, such that the final concentration of cell sample is plated in three 35-mm culture plates. The culture dishes are incubated at 37 º C at specific conditions of 5 % CO2, saturated humidity for a period of 14 days.

[0027] In a concluding step, 114, at the end of 7 days and 14 days, respective observations of the culture dishes of the CFU assay is conducted for assessment of repopulation capacity of the hematopoietic stem cells. Accordingly, Fig. 3A and 3B are representative of hematopoietic stem cell colonies after 7 days and 14 days of culture of CB mononuclear cells in the HSC CFU assay Media supplemented with the synergistic antibiotic composition

[0028] Various types of colonies are formed on the culture dish, the various types of colonies including erythroid progenitors, myeloid progenitors and multipotent colonies. Erythroid progenitors include colony-forming unit-erythroid and burst -forming unit-erythroid, the colony-forming unit-erythroid and burst -forming unit-erythroid representing the ability to produce colonies of maturing erythroblasts in the presence of erythropoietin. Further, myeloid progenitors include CFU-granulocyte, CFU-macrophage and CFU-granulocyte monocyte. More specifically, Figure 4 is representative of the CFU erythroid and BFU erythroid colonies of Hematopoietic stem cells.

[0029] The following examples are not intended to limit the scope of the disclosure. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0030] Example 1:

HSCs derived from a contaminated Human cord blood sample were suspended in a sterile optimized methylcellulose-based cell culture media at a final cell count of 2*104 cells. The microbial contamination included Pseudomonas aeruginosa, Staphylococcus aureus, E. coli and Acinetobacter baumanii (most common microbes encountered). An antibiotic stock solution of gentamicin at a concentration of 100 µg/ml was prepared and added to the methylcellulose-based culture media and the methylcellulose-based cell culture media is dispensed in culture dishes. The culture dishes are incubated at 37 º C at specific conditions of 5 % CO2, saturated humidity for a period of 14 days. The colonies were observed and classified by bright-field microscopy into the following categories based on size and morphology: CFU-E, BFU-E, CFU-GM, CFU-GEMM. The results observed denoted the growth of Acinetobacter baumanii and specific absence of growth of Pseudomonas aeruginosa, Staphylococcus aureus and E. coli.

Example 2

HSCs delivered from a contaminated Human cord blood sample were suspended in a sterile optimized methylcellulose-based cell culture media at a final cell count of 2*104 cells. The microbial contamination included specifically Pseudomonas aeruginosa, Staphylococcus aureus, E. coli and Acinetobacter baumanii microbes (most common microbes encountered). An antibiotic stock solution of a mixture of penicillin and streptomycin was prepared and added to the methylcellulose-based culture media and the methylcellulose-based culture media is dispensed in culture dishes. The culture dishes are incubated at 37 º C at specific conditions of 5 % CO2, saturated humidity for a period of 14 days. The colonies were observed and classified by bright-field microscopy into the following categories based on size and morphology: CFU-E, BFU-E, CFU-GM, CFU-GEMM. The results observed denoted the growth of Acinetobacter baumanii and absence of growth of Pseudomonas aeruginosa, Staphylococcus aureus and E. coli.

[0029] Example 3

HSCs delivered from a contaminated Human cord blood sample were suspended in a sterile optimized methylcellulose-based cell culture media at a final cell count of 2*104 cells. The microbial contamination included specifically Pseudomonas aeruginosa, Staphylococcus aureus, E. coli and Acinetobacter baumanii microbes (most common microbes encountered). An antibiotic stock solution of sulbactam at five different concentrations of 16 µg/ml, 30 µg/ml, 35 µg/ml, 40 µg/ml and 50 µg/ml were prepared and simultaneously a mixture of penicillin and streptomycin was prepared. Subsequently, five different aliquots of methylcellulose-based culture media were supplemented with a synergistic antibiotic composition including a mixture of penicillin and streptomycin and five different concentrations of 16 µg/ml, 30 µg/ml, 35 µg/ml, 40 µg/ml and 50 µg/ml respectively. Accordingly, the methylcellulose-based culture media was dispensed in culture dishes. The culture dishes were incubated at 37 º C at specific conditions of 5 % CO2, saturated humidity for a period of 14 days. The colonies were observed and classified by bright-field microscopy into the following categories based on size and morphology: CFU-E, BFU-E, CFU-GM, CFU-GEMM. The results observed denoted the growth of Staphylococcus aureus, E. coli at 16 µg/ml of sulbactam and growth of Pseudomonas aeruginosa at 30 µg/ml of sulbactam. The concentration range of 35-50 µg/ml of sulbactam in the antibiotic composition showed no growth of Pseudomonas aeruginosa, Staphylococcus aureus, E. coli and Acinetobacter baumanii and therefore complete control of microbial contamination was established.

[0031] Example 4

HSCs delivered from a contaminated Human cord blood sample were suspended in a sterile optimized methylcellulose-based cell culture media at a final cell count of 2*104 cells. The microbial contamination included specifically Pseudomonas aeruginosa, Staphylococcus aureus, E. coli and Acinetobacter baumanii microbes (most common microbes encountered). An antibiotic stock solution of sulbactam at a concentration of 50 µg/ml was added to the aliquot of methylcellulose-based culture media and the methylcellulose-based culture media was dispensed in culture dishes. The culture dishes were incubated at 37 º C at specific conditions of 5 % CO2, saturated humidity for a period of 14 days. The colonies were observed and classified by bright-field microscopy into the following categories based on size and morphology: CFU-E, BFU-E, CFU-GM, CFU-GEMM. The results observed denoted the growth of Pseudomonas aeruginosa, Staphylococcus aureus and absence of E. coli and Acinetobacter baumanii.

[0032] Table 1 summarizes the results of the inhibitory control against four specific microbes namely Pseudomonas aeruginosa, Staphylococcus aureus, E. coli and Acinetobacter baumanii.

Pseudomonas aeruginosa Staphylococcus aureus E. coli Acinetobacter baumanii
CONTROL (SAMPLE ONLY) BFU 0 0 0 0 0 0 0 0 0 0 0 0
CFU 0 0 0 0 0 0 0 0 0 0 0 0
BACTERIA growth +++ growth +++ growth +++ growth +++ growth +++ growth +++ growth +++ growth +++ growth +++ growth +++ growth +++ growth +++
SAMPLE + GENTAMYCIN BFU 3 2 4 2 4 2 2 2 3 2 1 1
CFU 4 6 5 1 1 2 4 6 2 1 2 1
BACTERIA no growth no growth no growth no growth no growth no growth no growth no growth no growth growth + growth + growth +
SAMPLE + PENSTREP (PS) BFU 4 2 2 2 1 4 4 3 5 0 0 0
CFU 4 3 4 2 3 1 5 9 2 0 0 0
BACTERIA no growth no growth no growth no growth no growth no growth no growth no growth no growth growth + growth + growth +
SAMPLE + PS + SULBACTUM 16(16 µg/ml) BFU 2 1 4 0 0 0 2 1 4 2 1 3
CFU 8 7 9 0 0 0 6 9 3 2 3 5
BACTERIA no growth no growth no growth growth + growth + growth + growth + growth + growth + no growth no growth no growth
SAMPLE + PS + SULBACTUM 30(30 µg/ml) BFU 2 1 3 0 0 0 2 5 1 4 7 2
CFU 5 4 5 2 1 1 4 8 2 6 8 11
BACTERIA growth + growth + growth + no growth no growth no growth no growth no growth no growth no growth no growth no growth
SAMPLE + PS + SULBACTUM 35(35µg/ml) BFU 3 2 5 3 6 2 4 5 3 3 3 3
CFU 5 3 7 4 2 8 8 7 6 2 5 3
BACTERIA no growth no growth no growth no growth no growth no growth no growth no growth no growth no growth no growth no growth
SAMPLE + PS + SULBACTUM 40(40µg/ml) BFU 1 1 1 5 7 4 3 7 2 5 7 3
CFU 4 3 5 2 1 3 4 2 7 9 10 6
BACTERIA no growth no growth no growth no growth no growth no growth no growth no growth no growth no growth no growth no growth
SAMPLE + PS + SULBACTUM 50(50 µg/ml) BFU 2 2 3 3 6 1 4 5 3 3 5 3
CFU 5 3 7 4 2 8 8 9 6 2 5 3
BACTERIA no growth no growth no growth no growth no growth no growth no growth no growth no growth no growth no growth no growth
SAMPLE + SULBACTUM 50(50 µg/ml) BFU 0 0 0 0 0 0 0 0 0 4 3 7
CFU 0 0 0 0 0 0 6 4 8 9 9 10
BACTERIA growth +++ growth +++ growth +++ growth +++ growth +++ growth +++ no growth no growth no growth no growth no growth no growth

[0033] In another embodiment, HSCs derived from a contaminated Human cord blood sample undergoes a routine CFU assay without the presence of antibiotics.

[0034] In a preferred embodiment, HSCs derived from a contaminated Human cord blood sample undergoes a sterility testing of the cord blood, without the presence of antibiotics prior to the CFU assay. Based on the type of microbial contamination encountered in the sterility test, an appropriate antibiotic composition is prepared to be added to the Methylcellulose-based CFU assay media for inhibiting microbial growth.

[0035] The present invention advantageously provides an antibiotic composition supplemented to the Methylcellulose-based CFU assay media for addressing a wide range of microbial contamination and promoting proliferation, growth and differentiation of HSCs in a CFU assay despite microbial contamination and further enabling selection of promising cord blood units for future storage as well as selection of one or more suitable units for transplantation.

[0036] The present invention further provides a ß-lactamase inhibitor acting as an adjuvant to the mixture of ß-lactam-Aminoglycoside antibiotics in a cell culture assay of a Hematopoietic stem cell CFU assay, thereby successfully blocking bacterial resistance mechanism and enabling growth and proliferation of hematopoietic stem cells.

[0037] Those skilled in the art will realize that the above recognized advantages and other advantages described herein are merely exemplary and are not meant to be a complete rendering of all of the advantages of the various embodiments of the invention.

[0038] In the foregoing specification, specific embodiments of the invention have been described. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the invention as set forth in the claims below. Accordingly, the specification and figures are to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the invention. The benefits, advantages, solutions to problems, and any element(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential features or elements of any or all the claims. The invention is defined solely by the appended claims including any amendments made during the pendency of this application and all equivalents of those claims as issued.

,CLAIMS:WE CLAIM:

1. A method of providing an optimized antimicrobial cell culture assay in a Hematopoietic stem cell CFU assay, the method comprising:
-supplementing a semi-solid CFU assay media with a synergistic antibiotic composition comprising one or more of a ß-lactamase inhibitor and a mixture of ß-lactam antibiotic and an Aminoglycoside to form a supplemented semi-solid based CFU assay media, wherein the synergistic antibiotic combination comprises one or more of a ß-lactamase inhibitor in a concentration range of 35-50 µg/ml and the mixture of ß-lactam antibiotic and Aminoglycoside comprising ß-lactam antibiotic in a concentration range of 50-200 I.U./ml and Aminoglycoside in a concentration amount of 50-200 µg/ml.

2. The method as claimed in claim 1, wherein the synergistic antibiotic composition comprises sulbactam and a mixture of penicillin and streptomycin.

3. The method as claimed in claim 2, wherein the synergistic antibiotic composition comprises sulbactam in a concentration range of 35-50 µg/ml.

4. The method as claimed in claim 2, wherein the synergistic antibiotic composition comprises a mixture of penicillin and streptomycin, wherein penicillin is supplemented in a concentration range of 50-200 I.U./ml and streptomycin is supplemented in a concentration amount of 50-200 µg/ml.

5. The method as claimed in claim 1, wherein the ß-lactam antibiotic is selected from a group of penicillin derivatives, cephalosporins, monobactams, and carbapenems.

6. The method as claimed in claim 1, wherein the Aminoglycoside is selected from a group of Actinomycin D, Kanamycin, Neomycin, Streptomycin and Gentamicin.

7. The method as claimed in claim 1, wherein the ß-lactamase inhibitor is selected from
a group of sulbactam, clavulanate, tazobactam and avibactam.

8. The method as claimed in claim 1, wherein the semi-solid CFU assay media is a Methylcellulose-based CFU assay media.

9. The method as claimed in claim 8, wherein the Methylcellulose-based CFU assay media further comprises methylcellulose, fetal bovine serum, bovine serum albumin, L-glutamine, a reducing agent, a basal medium and a plurality of recombinant cytokines.

10. The method as claimed in claim 1, wherein the synergistic antibiotic composition comprises a ß-lactamase inhibitor to form an optimized Methylcellulose-based CFU assay media.

11. The method as claimed in claim 10, wherein the synergistic antibiotic composition comprising a ß-lactamase inhibitor in a concentration of 50 µg/ml is effective against a group of gram negative bacteria.

12. The method as claimed in claim 11, wherein the synergistic antibiotic composition comprising a ß-lactamase inhibitor in a concentration of 50 µg/ml is effective against one or more of gram negative bacteria selected from Escherichia coli, Acinetobacter baumanii and the likes thereof.

13. The method as claimed in claim 1, wherein the microbial contaminants in harvested human umbilical cord blood sample comprise a plurality of Gram positive bacteria.

14. The method as claimed in claim 1, wherein the microbial contaminants in harvested human umbilical cord blood sample comprise a plurality of Gram negative bacteria.

15. The method as claimed in claim 1 further comprises performing a qualitative test to analyze the potency of HSCs isolated from human umbilical cord blood cells.