DESC:FIELD OF THE INVENTION:
This invention relates to the field of biomedical engineering.

Particularly, this invention relates to the field of tissue engineering.

Specifically, this invention relates to a bio-engineered skin tissue device for cosmetic screening and / or drug screening applications.

BACKGROUND OF THE INVENTION:
Bioengineered skin models are desirable models for testing and screening of dermatological drugs, formulations, cosmetics, and other such items / applications. They also find applications in graft / implant applications for patients with burns and skin wounds. There are several skin models developed so far through different approaches.

However, there are several deficiencies in the prior art when it comes to in-vitro and in-vivo analysis using such models.

OBJECTS OF THE INVENTION:
An object of the invention is to provide an efficient and cost-effective device for in-vitro and in-vivo analysis in clinical trial of drugs and cosmetic testing.

Another object of the invention is to relatively reduce huge amounts of money and animal sacrifices for cosmetic screening.

Yet another object of the invention is to provide an easy-to-use technology and method for chemical trials on human skin.

Still another object of the invention is to eliminate need of animals and cadavers skin during cosmetic screening.

Another additional object of the invention is to eliminate risk of non-compatible, irritating and harmful chemical in cosmetic products.

Yet another object of the invention is to accurately compatible product through bio-engineered skin tissue.

SUMMARY OF THE INVENTION:
According to this invention, there is provided a bio-engineered skin tissue device for cosmetic screening and / or drug screening applications, said device comprising:
- a bottom layer being a filtrate chamber comprising a filter and sealed off with a chamber cover such that culture media from a media chamber, positioned above it, is filtered by the filter and becomes recycled media which is stored in the bottom layer;
- a pump connected to the bottom layer such that said pump takes the recycled media, from the bottom layer, and sends it to the media chamber;
- a syringe connected to a pipe connected to the filter such that the syringe takes the culture media, from the media chamber, and sends it, with the help of a pipe, connected to the filter;
- a culture plate, positioned operatively atop said media chamber, such that the media chamber is configured to be connected to the syringe; and
- a supporting plate, located operatively atop the culture plate, the supporting plate connected to the media chamber such that the culture plate hangs on to the media chamber and the culture plate has pores where media passes and reaches to cells on the culture plate.

In at least an embodiment, said device includes a cap covering the entire assembly of layers below it.

In at least an embodiment, said filter being sterilizing-grade filters having the pore size of 01.-0.2um.

In at least an embodiment, said culture plate is a macro porous support which houses a multi-scale scaffold having nanofibrous matrix integrated with microporous gel, said culture plate enabling growth of two different cell types.

In at least an embodiment, said device comprising:
- a bottom layer, atop said supporting plate, said bottom layer comprising gel over on fibroblast cell; and
- a top layer, atop said bottom layer, comprising nanofiber over housing Keratinocytes.

In at least an embodiment, said device includes an exchange chamber as the bottom layer and a nanofibrous scaffold on top of it for cell culture with its next layer of cell seeded in the form of a bioink, both the layers being irrigated with the nutrient and gas mixture present in the exchange chamber.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
The invention will now be described in relation to the accompanying drawings, in which:
FIGURE 1 illustrates the bio-engineered skin tissue device (100) of this invention; assembled, with all components;
FIGURE 2 illustrates an exploded view, showing layers, of the bio-engineered skin tissue device of this invention;
FIGURE 3 illustrates the description of each component of the bio-engineered skin device;
FIGURE 4a illustrates protocol steps for Hydrogel development in the bio-engineered skin device of Figures 1 and 2;
FIGURE 4b illustrates protocol steps for Nanofiber development in the bio-engineered skin device of Figures 1 and 2;
FIGURE 5 illustrates printing of Hydrogel and deposition of Nanofiber, on the culture plate, of the bio-engineered skin device of Figures 1 and 2;
FIGURE 6 illustrates the scanning electron micrograph (SEM) of the electrospun PCL-Gel nanofiber matrix showing its morphology (left) and contact angle measure on electrospun PCL-Gel nanofiber matrix showing its wettability nature;
FIGURE 7 illustrates 7(1) image showing bioprintability of the bionink (gelatin gel) developed 7(2) image showing the layer stacking of the developed gelatin gel during bioprinting 7(3) image showing the pore geometry of the 3D bioprinted structure with the developed gelatin gel 7(4) scanning electron micrograph of the lyophilized sample of the developed gelatin gel 7(5) graph showing viscosity analysis of the gelatin gel 7(6) graph showing swelling studies of gelatin gel in buffer solution 7(7) graph showing the degradation behaviour of the gelatin gel in buffer solution;
FIGURE 8 illustrates assembly of the functional unit of bioengineered skin device with SEM images of multiscale architecture achieved through integration of nanofber mat and gelatin gel layer. SEM images illustrate the microstructure at different magnification of the developed multiscale composite scaffold which forms the functional unit of cell culture plate in the bioengineered skin device;
FIGURE 9 illustrates: (a) SEM Image of Polycaprolactone(PCL) and Gelatin(Ge) Electrospun Fibre, fluorescent microscope images of L929 fibroblast cell line dyed with (b) FAD, (c) PID, (d) merged (b, c and d are for Day – 1), (e) FAD, (f) PI and (g) merged (e, f, and g are for Day – 4). All the results have been observed with L929 fibroblast cell line; and
FIGURE 10 illustrates: (a) SEM Image of Gelatin(Ge) & Sodium Alginate(SA) gel, fluorescent microscope images of MG-63 cell line dyed with (b) Acridine orange, (c) ORO, (d) merged (b, c and d are for Day – 1), (e) Acridine orange, (f) ORO and (g) merged (e, f and g are for Day – 4). All the results have been observed with MG-63 cell line).

DETAILED DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
According to this invention, there is provided a bio-engineered skin tissue device for cosmetic screening and / or drug screening applications.

This invention relates to systems and methods for cosmetic testing and neo drug analysis based on tissue engineering in relation to research and clinical applications. The device, of this invention, supports development of a multiscale scaffold architecture having integrated properties of hydrogel and nanofibers.

FIGURE 1 illustrates the bio-engineered skin tissue device (100) of this invention; assembled, with all components.

The proposed device, of this invention, is a miniaturized biochemical reactor that is capable of:
? Maintaining continuous and controlled supply of nutrients and gas;
? Maintaining controlled temperature environment;
? Providing morphological support structure;
Necessary to support cell growth and multiplication to form functional tissue comprising multilayered cellular structures.

FIGURE 2 illustrates an exploded view, showing layers, of the bio-engineered skin tissue device of this invention.

In at least an embodiment, the device comprises a bottom layer (12) being a filtrate chamber comprising a filter (11) and sealed off with a chamber cover (10). Typically, the culture media come from the Media chamber (6) and filter the (Excretory product Ex. urea etc.) by filter (11) and and become recycled media which stored in the filter chamber (12)

In at least an embodiment, the device comprises a pump (9) connected to the bottom layer (12). Pump (11) takes the recycled media from the filtrate chamber and sends it to the media chamber.

In at least an embodiment, the device comprises a syringe (7) connected to a pipe (8) connected to the filter (11). Typically, the syringe (7) takes the culture media from media chamber (6) and send it with the help of pipe (8) to the filter (11).

In at least an embodiment, the device comprises a media chamber holding a culture plate (5), the media chamber being configured to be connected to the syringe (7). A supporting plate (4) is located atop the culture plate (5).

The culture plate (5) is attached to the supporting plate (4) and the Supporting plate (4) is attached to the media chamber (6). So, in this arrangement, the culture plate (5) hangs on media chamber and the culture plate have some pores where media pass and reach to the cells.

In at least an embodiment, the device comprises a first layer (3)comprising gel over on fibroblast cell. Fibroblast cells require a specific type of environment in order to effectively proliferate and form a dermis layer. This gel is specifically designed to mimic the natural matrix found in the body, providing an ideal environment for fibroblast cells to grow and thrive. By using a gel matrix, to create a controlled environment that closely mimics the natural conditions found in the body. This allows them to study fibroblast cell behaviour in a more accurate and relevant way, and can help to inform the development of new treatments and therapies for a range of conditions.

In at least an embodiment, the device comprises a second layer (2) comprising nanofiber over on Keratinocytes. The morphology and texture of nanofibrous layer may be tailored to suit the needs of different kind of cells. Keratinocytes are the primary cells found in the epidermis, which is the outermost layer of the skin. This skin layer serves as a protective barrier against external factors such as UV radiation, pollutants, and pathogens. When keratinocytes are grown on nanofibers, they tend to align themselves along the fibers' orientation. This alignment enables the cells to form a more organized and uniform layer, similar to the epidermis's structure. The nanofibers' rough surface also provides mechanical cues to the keratinocytes, which stimulate their proliferation and migration. The unique properties of nanofibers, including their high surface area and porosity, make them an ideal material for creating a dermis-epidermis junction.

In at least an embodiment, the device comprises a cap (1) covering the entire assembly of layers below it.

The device comprises an exchange chamber as the bottom layer and a nanofibrous scaffold on top of it for cell culture. The next layer of cell is seeded in the form of a bioink. Both the layers are irrigated with the nutrient and gas mixture present in the exchange chamber.

The scaffold and device design acts as a perfusion bioreactor where air-liquid interface is maintained through irrigating the scaffold from below and effective mass transport can be achieved by continuous circulation of media.

The invention enables the culture of two different cell types as observed in commercial insert and well plate assembly. For instance, keratinocytes are grown on nanofibrous top layer and fibroblast in microporous gel layer.

Any polymers, that can be converted to nanofibers through electrospinning and bioink for bioprinting, can be integrated through the proposed method of this invention. Owing to the different polymers being utilised, gradient stiffness of the scaffold can be achieved through tailoring material type and porosity level. The morphological features of scaffold can be tailored as per the requirement. The pore connectivity in the scaffold support effective transport of nutrients and waste. The multiscale scaffold supports the sequential seeding of cells to form two-layered tissue sheets.

In preferred embodiments, the multiscale scaffold developed shows the nanofiber diameter (200-800nm) and pore size (2 - 200um).

In preferred embodiments, cells can be grown in or on a membrane of thickness 200um to form a sheet of cells having 3.5mm diameter, apt for mimicking barrier function of tissues like skin, cornea, intestine, lungs, and others.

FIGURE 3 illustrates the description of each component of the bio-engineered skin device

FIGURE 4a illustrates protocol steps for Hydrogel development in the bio-engineered skin device of Figures 1 and 2.
FIGURE 4b illustrates protocol steps for Nanofiber development in the bio-engineered skin device of Figures 1 and 2.

FIGURE 5 illustrates printing of Hydrogel and deposition of Nanofiber, on the culture plate, of the bio-engineered skin device of Figures 1 and 2.
STEP 401: Gelatin Gel Composition: 10% wt./vol. Gelatin + 1% wt./vol. Sodium Alginate; both added in water
STEP 402: mixer was put on magnetic stirrer for 30 min. at 37 OC
STEP 403: solution is kept in sonicator for 30 min. at 50 OC
STEP 404: after sonication, add 2% CaCl2 in solution and mix well
STEP 405: then, solution is immediately loaded in syringe
STEP 406: after some time, gel is formed; Gel is ready for printing
STEP 407: output is gel-loaded syringe

STEP 501: Nanofiber Gelatin Composition: 12% vol./vol. Gelatin + 12% vol./vol. PCL + 50µL in 1 mil solution; all dissolved in HFIP
STEP 502: solution is kept on magnetic stirrer at room temperature for 24 hours
STEP 503: solution is ready for Electrospinnin
STEP 504: Output is Nanofiber layer

The device, of this invention, comprises a proprietary 3-dimensional bio-engineered skin tissue which is designed to facilitate ease of continuously flowing media supplement, storage, and support easy transport to cosmetic / pharma companies and research laboratory without contamination and biohazard problems. However, the prior 2D cell culture-based tissue devices and methods are complex in handling and also not control of cell growth and culture process. The invented device enables the collection of accurate and precise skin tissue from 3D Bio-engineering technology for cosmetic/drug screening.

The proposed reactor is suitable for evaluating drug testing (permeability) through the developed cell sheet membrane.

The device can be disassembled easily for easy Imaging based characterization of the developed tissue

FIGURE 6 illustrates the scanning electron micrograph (SEM) of the electrospun PCL-Gel nanofiber matrix showing its morphology (left) and contact angle measure on electrospun PCL-Gel nanofiber matrix showing its wettability nature.
FIGURE 7 illustrates 7(1) image showing bioprintability of the bionink (gelatin gel) developed 7(2) image showing the layer stacking of the developed gelatin gel during bioprinting 7(3) image showing the pore geometry of the 3D bioprinted structure with the developed gelatin gel 7(4) scanning electron micrograph of the lyophilized sample of the developed gelatin gel 7(5) graph showing viscosity analysis of the gelatin gel 7(6) graph showing swelling studies of gelatin gel in buffer solution 7(7) graph showing the degradation behaviour of the gelatin gel in buffer solution
FIGURE 8 illustrates assembly of the functional unit of bioengineered skin device with SEM images of multiscale architecture achieved through integration of nanofber mat and gelatin gel layer. SEM images illustrate the microstructure at different magnification of the developed multiscale composite scaffold which forms the functional unit of cell culture plate in the bioengineered skin device.

FIGURE 9 illustrates: (a) SEM Image of Polycaprolactone(PCL) and Gelatin(Ge) Electrospun Fibre, fluorescent microscope images of L929 fibroblast cell line dyed with (b) FAD, (c) PID, (d) merged (b, c and d are for Day – 1), (e) FAD, (f) PI and (g) merged (e, f, and g are for Day – 4). All the results have been observed with L929 fibroblast cell line

FIGURE 10 illustrates the MG-63 cells printed with a bioink made up of gelatine (10 wt%) and sodium alginate (4 wt%) on day – 1 and day – 4.
FIGURE 10 illustrates: (a) SEM Image of Gelatin(Ge) & Sodium Alginate(SA) gel, fluorescent microscope images of MG-63 cell line dyed with (b) Acridine orange, (c) ORO, (d) merged (b, c and d are for Day – 1), (e) Acridine orange, (f) ORO and (g) merged (e, f and g are for Day – 4). All the results have been observed with MG-63 cell line).

According to non-limiting exemplary embodiment, the inventors have performed experiments to establish that co-culture of cells is possible in the perfusion reactor. The images in figures 5 and 6 show the results of cells cultured on two layers (one nanofibrous and one printed gel layer). The figures show the microscopy images from day-1 and day-4 also, which establishes that cells not only grow but also remain viable in both the layers over a period of 4 days.

Experiments were performed to establish the cell proliferation and cell viability with two different types of cell lines, L929 (adherent mouse fibroblast cell lines) and MG-63 (osteosarcoma cell lines). The former was used for studies on nanofibrous substrate, while the latter was used to form the ink and was printed using the bio-printer.

Both the experiments have shown significant viability and proliferation on both the substrates. Two different cell-lines were used as this is what was originally proposed – culturing two different cell lines in gel and nanofibrous substrate. I am showing the results in figures below. Figure 1 shows the L929 cells cultured on PCL (2 wt%) and gelatin (8 wt%) nanofibers, on day –1 and day – 4.

The TECHNICAL ADVANCEMENT of this invention lies in providing method of seeding and supporting two layers of cells of different kinds.

While this detailed description has disclosed certain specific embodiments for illustrative purposes, various modifications will be apparent to those skilled in the art which do not constitute departures from the spirit and scope of the invention as defined in the following claims, and it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.
,CLAIMS:WE CLAIM,

1. A bio-engineered skin tissue device for cosmetic screening and / or drug screening applications, said device comprising:
- a bottom layer (12) being a filtrate chamber comprising a filter (11) and sealed off with a chamber cover (10) such that culture media from a media chamber (6), positioned above it, is filtered by the filter (11) and becomes recycled media which is stored in the bottom layer (12);
- a pump (9) connected to the bottom layer (12) such that said pump (9) takes the recycled media, from the bottom layer (12), and sends it to the media chamber (6);
- a syringe (7) connected to a pipe (8) connected to the filter (11) such that the syringe (7) takes the culture media, from the media chamber (6), and sends it, with the help of a pipe (8), connected to the filter (11);
- a culture plate (5), positioned operatively atop said media chamber (6), such that the media chamber (6) is configured to be connected to the syringe (7); and
- a supporting plate (4), located operatively atop the culture plate (5), the supporting plate (4) connected to the media chamber (6) such that the culture plate (5) hangs on to the media chamber (6) and the culture plate (5) has pores where media passes and reaches to cells on the culture plate (5).

2. The device as claimed in claim 1 wherein, said device includes a cap (1) covering the entire assembly of layers below it.

3. The device as claimed in claim 1 wherein, said filter (11) being sterilizing-grade filters having the pore size of 01.-0.2um.

4. The device as claimed in claim 1 wherein, said culture plate (5) is a macro porous support which houses a multi-scale scaffold having nanofibrous matrix integrated with microporous gel, said culture plate (5) enabling growth of two different cell types.

5. The device as claimed in claim 1 wherein, said device comprising:
- a bottom layer (3), atop said supporting plate (4), said bottom layer (3) comprising gel over on fibroblast cell; and
- a top layer (2), atop said bottom layer (3), comprising nanofiber over housing Keratinocytes.

6. The device as claimed in claim 1 wherein, said device includes an exchange chamber as the bottom layer and a nanofibrous scaffold on top of it for cell culture with its next layer of cell seeded in the form of a bioink, both the layers being irrigated with the nutrient and gas mixture present in the exchange chamber.