The present disclosure relates to cryopreservation composition. In particular, a cryopreservation composition for preserving cells and corresponding methods are provided. The said cryopreservation composition is an improved and efficient formulation which offers several advantages. Further, the cryopreserved cells show excellent viability, quality, stability and biological functionality over prolonged periods. BACKGROUND OF THE DISCLOSURE

Cryopreservation is a process where biological samples such as cells or whole tissue are preserved by cooling to low sub-zero temperature. Preservation of cells (especially stem cells) is critical for both research and clinical application of stem-cell based therapies. Preservation permits development of cell banks with stem cells from various sources such as bone marrow, umbilical cord, cord blood, dental pulp and adipose tissue. The ability to preserve cells permits the banking of stem cells until later use in research lab or clinical application. Further, the ability to preserve cells permit completion of quality and safety testing before use as well as transportation of cells between the sites of collection, processing and clinical administration. Finally, preservation of cells therapeutically facilitates the development of a manufacturing paradigm for stem cell based therapies. Hence, preservation of cells (especially stem cells) is an important aspect due to its wide range of implications which it possesses.

The immunomodulatory and tissue regenerative properties of mesenchymal stem cells (MSCs) make them attractive therapeutic modality for treating a wide range of diseases. However, highly demanding cell doses used in MSC clinical trials (millions of cells/kg body weight) require freezing and storage conditions without impacting the viability and multipotency of the frozen cells for prolonged duration. Currently, practices of freezing cells require extensive post thaw manipulation like reconstitution, dilutions, centrifugations which have a risk of impact on viability, cell loss during transplantation or infusion. Therefore, it is necessary to maintain and retain all functional properties of stem cells for therapeutic applications. In addition, cryopreserving large batches of stem cells is one of the major challenges and is highly a time dependent process as encountered by the currently practiced methods. Further, effective and efficacious therapeutic advantage of stem cells can be accelerated, maintained and translated into the clinical application by effective cryopreservation of stem cells. Towards this, the currently available methods employ bio-preservation media which plays an important role to maintain stability, increase shelf life period to bring and preserve stem cells in a stable, non-manipulated ready to use format without losing any functional characteristics.

However, conventional Plasmalyte A based cryopreservation formulations which are currently in practice contain higher percentage of dimethyl sulfoxide (DMSO) [such as 10% DMSO or more] which enhances the lethality associated with cooling and thawing processes. Many adverse effects and toxic reactions are evident in many patients treated with cells frozen in high DMSO concentration in clinical settings. Hence, it is necessary to reduce the toxicity during such cases by diluting or washing or completely removing the DMSO from cryopreserved cells before practical applications (such as administration of cells to patient). Also, these post thaw manipulations of cryopreserved stem cell product further decrease the viability, are time consuming and require aseptic zone of processing and concentrating cells at the site.

To overcome post thaw manipulations (like reconstitution, centrifugation, concentration and making pre filled syringes) and to achieve improved characteristics such as better cell recovery, viability & long term stability without compromising phenotype and multipotency of cells, there is a need to develop efficient compositions and related methods. Accordingly, the present disclosure aims to address the aforesaid limitations of the prior art by providing efficient cryopreservation/freezing compositions and corresponding methods.

STATEMENT OF THE DISCLOSURE

Accordingly, the present disclosure relates to a cryopreservation composition comprising ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agents; a method of cryopreservation of cells comprising step of suspending the cells in said cryopreservation composition and a method for maintaining viability and stability of cells comprising step of suspending the cells in the cryopreservation composition. BRIEF DESCRI PTION OF THE ACCOMPANYING FIGURES

In order that the invention may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated and the accompanying figures. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present invention wherein:

Figure 1 depicts viability of cryopreserved mesenchymal stem cells (MSCs) by die exclusion method for different freezing densities with respect to different time points.

Figure 2 depicts viability of cryopreserved MSCs by flow cytometer using 7AAD at 12 week i.e. time point 5.

Figure 3 depicts total cell recovery from different freezing densities with respect to different time points.

Figure 4 depicts the day three (03) morphology of cultures derived from frozen-thawed MSCs wherein the MSCs are frozen till time point 5 at different freezing densities (image 10X) viz. 5 million cells/ml, 10 million cells/ml, and 15 million cells/ml respectively.

Figure 5 depicts the colony forming unit (CFU-F) assay at time point 5 for MSCs frozen at different freezing density. Figure 6 depicts the qualitative CFU-F efficacy of frozen cells at different freezing densities - 5 million cells/ml, 10 million cells/ml and 15 million cells/ml at time point 5. The cells are stained with India ink to show small and big CFU-F/well appearance (Figure 6A). The figure also provides graphical representation of the CFU-F assay for MSCs frozen at different freezing densities viz. 5 million cells/ml, 10 million cells/ml and 15 million cells/ml frozen cells at time point 5 (Figure 6B).

Figure 7 depicts the results of Adipocyte differentiation assay. Morphology of differentiating adipocytes [Figure 7A: (a) 5 million cells/ml frozen cells; (b) 10 million cells/ml frozen cells and (c) 15 million cells/ml frozen cells] and the quantification results are shown at time point 5 for MSCs frozen at different freezing densities (Figure 7B). Figure 8 depicts the results of Osteogenesis differentiation assay. Morphology of differentiating osteocytes [Figure 8 (a) 5 million cells/ml frozen cells; (b) 10 million cells/ml frozen cells and (c) 15 million cells/ml frozen cells] are shown at time point 5 for MSCs frozen at different freezing densities.

Figure 9 depicts the results of Chondrogenesis differentiation assay. Morphology of differentiating chondrocytes [Figure 9A(a) 5 million cells/ml frozen cells; (b) 10 million cells/ml frozen cells and (c) 15 million cells/ml frozen cells] and its quantification results (Figure 9B) are shown at time point 5 for MSCs frozen at different freezing densities.

Figure 10 depicts the results for VEGF potency assay at 48 and 72 hours respectively using the conditioned medium from frozen-thawed MSCs at different freezing densities for 12 weeks (time point 5). DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to a cryopreservation composition comprising ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agents.

In an embodiment of the disclosure, the ionic buffer is selected from a group comprising PlasmaLyte A, ringer lactate solution, sucrose and combinations thereof. In another embodiment, the cryoprotectant is selected from a group comprising dimethylsulfoxide, glycerol, triglycerol and combinations thereof.

In yet another embodiment, the protein is selected from a group comprising human serum albumin, recombinant plant derived xenofree human serum albumin and a combination thereof. In a further embodiment, the energy substrate is selected from a group comprising trehalose, hydroethyl starch and a combination thereof.

In a still further embodiment, the anti-aging agent is a combination of L-Glutamine, poly-L- lysine and ectoine.

In still another embodiment, the ionic buffer is present at a concentration ranging from about 80% to 90% v/v; the cryoprotectant is present at a concentration ranging from about 3% to 10%) v/v; the protein component is present at a concentration ranging about 3% to 7.5% v/v; the energy substrate is present at a concentration ranging from about 0.25% to 2.50% v/v; and the anti-aging agent is present at a concentration ranging from about 0.0005% to 2% v/v. In still another embodiment, said cryopreservation composition is xenofree and is for cryopreservation of cells.

The present disclosure further relates to a method of cryopreservation of cells comprising step of suspending the cells in the cryopreservation composition.

The present disclosure also relates to a method for maintaining viability and stability of cells comprising step of suspending the cells in the cryopreservation composition.

In an embodiment of the present disclosure, the cells are selected from a group comprising stem cells, human cells, animal cells, plant cells and combinations thereof. In a preferable embodiment, the cells are stem cells.

In yet another embodiment, the cell s are suspended at a freezing density ranging from about 5 million cells per ml to 30 million cells per ml of the composition.

In still another embodiment, viability of the cells is maintained at a range from about 70%> to 100 %.

In a still further embodiment, the cells can be cryopreserved for a time-period ranging from about 1 day to 36 weeks. In another embodiment, the cells can be cryopreserved for a time-period ranging from 1 day to 12 weeks.

In yet another embodiment, the cryopreserved cells retain differentiation potential. In still another embodiment, stability of cryopreserved cells post thawing is maintained for a time-period ranging from about at least 2 hours to 5 hours at a temperature ranging from about 21° C to 25° C. In another embodiment, the cryopreserved cells can be directly administered to a subject in need thereof without any post thaw manipulation.

As used herein, the terms "cryopreservation media", "cryopreservation composition", "cryopreservation formulation", "cryopreservation solution", "freezing composition", "freezing formulation" and "freezing media" are used interchangeably within this disclosure which refer to the product of the disclosure.

One of the objectives of the instant disclosure is to provide for efficient cryopreservation compositions and methods which minimize damage to cells during low temperature freezing and storage. The detrimental effects of cellular cryopreservation is minimized by controlling the cooling rate, using better cryoprotective agent(s), maintaining appropriate storage temperatures, and controlling the cell thawing rate. Accordingly, in the present disclosure, clinical grade cryopreservation media for cryopreserving stem cells at high freezing density for prolonged periods is developed and critically evaluated. Quantitative tests such as cell viability, immunophenotype, multipotential differentiation capacity and analysis of presence of apoptotic cells prove that these characteristics in frozen stem cells are not altered. Further, enhanced proliferation rates of post-cryopreserved stem cells are observed.

The present disclosure provides for a xenofree 'cryopreservation media' with low concentration of cryoprotecting agent to freeze the cell therapy product. Furthermore, said composition/media supports the preservation of stem cells (especially MSC's) at high concentrations. In an embodiment, when the cells are preserved at a concentration/freezing density of greater than 25 million cells per ml, the cryopreserved cells show good stability during long term storage.

The cryopreservation/freezing composition of the instant disclosure comprises ionic balance buffer, cryoprotectant/cryopreservation antifreeze protectant, a protein component, energy substrate and anti-ageing agent. In an embodiment of the present disclosure, the ionic buffer is selected from a group comprising but not limiting to PlasmaLyte A [defined as Multiple Electrolytes Injection, Type I, USP comprising Sodium Chloride, Sodium Gluconate, Sodium Acetate Trihydrate, Potassium Chloride, and Magnesium Chloride], Ringer Lactate solution, sucrose and combinations thereof. In an embodiment of the present disclosure, the cryopreservation antifreeze protectant is either permeating or non-permeating in nature.

In another embodiment of the present disclosure, the cryopreservation antifreeze protectant is selected from a group comprising but not limiting to dimethyl sulfoxide (DMSO), glycerol, triglycerol or any combination thereof.

In a preferable embodiment of the present disclosure, low concentration of DMSO in combination with glycerol is employed as cryopreservation antifreeze protectant which helps in achieving significant success of freezing and avoiding toxicity.

In another embodiment of the present disclosure, the protein component is selected from a group comprising but not limiting to human serum albumin (HSA), cellastim (a recombinant plant derived xenofree HSA), or a combination thereof.

In another embodiment of the present disclosure, the energy substrate is selected from a group comprising but not limiting to Trehalose, Hydroxyethyl Starch, or a combination thereof. In another embodiment of the present disclosure, the anti-ageing agent is a combination of L- Glutamine, Poly-L-Lysine and Ectoine.

In an embodiment of the present disclosure, the concentration [volume/volume (v/v)] of various components of the cryopreservation composition is as follows: ionic buffer ranges from about 80 % v/v to 90 % v/v, preferably about 80 % v/v to 85 % v/v; cryopreservation antifreeze protectant ranges from about 3 % v/v to 10 % v/v, preferably about 4 % v/v to 5% v/v; protein component ranges from about 3 % v/v to 7.5 % v/v, preferably about 4 % v/v to 5% v/v; energy substrate ranges from about 0.25% v/v to 2.50 % v/v, preferably about 0.75% % v/v to 1.50 % v/v, and anti-ageing agent ranges from about 0.0005 % v/v to 2 % v/v, preferably about 1 % v/v to 2 % v/v.

The cryopreservation composition of the present disclosure contains low concentration of dimethylsulfoxide. In particular, the concentration of dimethylsulfoxide in the cryopreservation composition does not exceed 5% v/v. The components of the instant cryopreservation composition have specific roles/functions which are important for effective cryopreservation of the cells. The roles/functions of the components are described in the table below.

Table 1 : Roles/functions of the components of the composition

In an exemplary embodiment of the present disclosure, a cryopreservation composition comprising DMSO, Glycerol, Human Serum Albumin, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L-Lysine, Ectoine and PlasmaLyte A is provided. In another embodiment of the present disclosure, a cryopreservation composition comprising Ringer Lactate solution, Glycerol, Human Serum Albumin, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L- Lysine, Ectoine and PlasmaLyte A is provided. In yet another embodiment of the present disclosure, a cryopreservation composition comprising Sucrose, Glycerol, Human Serum Albumin, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L- Lysine, Ectoine and PlasmaLyte A is provided.

In still another embodiment of the present disclosure, a cryopreservation composition comprising DMSO, Glycerol, Cellastim, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L- Lysine, Ectoine and PlasmaLyte A is provided.

In yet another embodiment of the present disclosure, a cryopreservation composition comprising DMSO, Glycerol, Human Serum Albumin, Hydroxyethyl starch, GlutaMax, Poly-L-Lysine, Ectoine and PlasmaLyte A is provided.

In yet another embodiment of the present disclosure, a cryopreservation composition comprising DMSO, Glycerol, Human Serum Albumin, Trehalose, GlutaMax, Poly-L-Lysine, Ectoine and PlasmaLyte A is provided.

In another embodiment of the present disclosure, a cryopreservation composition comprising Ringer Lactate solution, Glycerol, Cellastim, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L-Lysine, Ectoine and PlasmaLyte A is provided. In a still further embodiment of the present disclosure, a cryopreservation composition comprising DMSO, Glycerol, Human Serum Albumin, Trehalose, Hydroxyethyl starch, GlutaMax, Poly-L-Lysine, Ectoine and PlasmaLyte A is provided.

In exemplary embodiments, the concentration of the components of the above cryopreservation compositions is provided. In an embodiment, the concentration (v/v) of ionic buffer selected from a group comprising PlasmaLyte A, Ringer Lactate solution, sucrose and combinations thereof is selected from 80 %, 81 %, 82 %, , 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 % or 90 % v/v. In another embodiment, the concentration (v/v) of cryoprotectant/ cryopreservation antifreeze protectant selected from a group comprising dimethyl sulfoxide (DMSO), glycerol, triglycerol and combinations thereof is selected from 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% v/v.

In still another embodiment, the concentration (v/v) of protein component selected from a group comprising human serum albumin, recombinant plant derived xenofree human serum albumin, and a combination thereof is selected from 3%, 4%, 5%, 6%, 7% or 7.5% v/v. In yet another embodiment, the concentration (v/v) of energy substrate selected from a group comprising Trehalose, Hydroxyethyl Starch, or a combination thereof is selected from 0.25%, 1.25%, 2.25% or 2.5% v/v.

In a further embodiment, the concentration (v/v) of anti-aging agent - a combination of L- Glutamine, Poly-L-Lysine and Ectoine s selected from 0.0005%, 1.0005% or 2% v/v.

In a still further embodiment, the concentration (v/v) of ionic buffer selected from a group comprising PlasmaLyte A, Ringer Lactate solution, sucrose and combinations thereof ranges from about 80% to 85% v/v.

In another embodiment, the concentration (v/v) of cryoprotectant/ cryopreservation antifreeze protectant selected from a group comprising dimethyl sulfoxide (DMSO), glycerol, triglycerol and combinations thereof ranges from about 4%> to 5% v/v. In still another embodiment, the concentration (v/v) of protein component selected from a group comprising human serum albumin, recombinant plant derived xenofree human serum albumin, and a combination thereof ranges from about 4% to 5% v/v.

In yet another embodiment, the concentration (v/v) of energy substrate selected from a group comprising Trehalose, Hydroxyethyl Starch, or a combination thereof ranges from about 0.75% to 1.50% v/v.

In a further embodiment, the concentration (v/v) of anti-aging agent comprising a combination of L-Glutamine, Poly-L-Lysine and Ectoine ranges from about 1%> to 2% v/v. In an exemplary embodiment, all cells types from various sources can be cryopreserved in the cryopreservation composition of the instant disclosure. In a preferred embodiment, the cell type is stem cells. In a specific embodiment, the cells are mesenchymal stem cells (MSCs). Further, said stem cells can be obtained from sources selected from a group comprising bone marrow, umbilical cord, blood, dental pulp and adipose tissue. In a specific embodiment, the source of stem cells is bone marrow.

In an embodiment of the present disclosure, the cryopreservation composition of the instant disclosure comprises United States Pharmacopeia (USP) grade components/reagents.

The present disclosure demonstrates that human MSCs can be successfully cryopreserved at high freezing density for banking and clinical applications. A freezing density ranging from about 15 million cells/ml to about 30 million cells/ml, more preferably about 15 million cells/ml to about 25 million cells/ml is achieved by the cryopreservation composition of the instant disclosure. In an embodiment of the present disclosure, the frozen MSCs can be delivered to the bedside clinically.

In specific embodiments, the freezing density (no. of cells/ml of the cryopreservation composition) of human stem cells such as MSCs is 15 million cells/ml, 16 million cells/ml, 17 million cells/ml, 18 million cells/ml, 19 million cells/ml, 20 million cells/ml, 21 million cells/ml, 22 million cells/ml, 23 million cells/ml, 24 million cells/ml, 25 million cells/ml, 26 million cells/ml, 27 million cells/ml, 28 million cells/ml, 29 million cells/ml or 30 million cells/ml. In an embodiment, the present disclosure provides for a xenofree cryopreservation formulation for enhanced/excellent cell viability and functionality while eliminating the need for high levels of cytotoxic agents.

In another embodiment of the present disclosure, at least about 95% of frozen cells are recovered as live cells after freezing in the instant formulation followed by storage in liquid nitrogen for about 12 weeks. In other words, cell recovery after 12 weeks storage is about 95% or greater. These results establish that the cells frozen in the instant formulation retain excellent cell viability even at longer storage periods. In yet another embodiment of the present disclosure, the cell viability of the cells cryopreserved in the instant formulation is selected from 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%. In preferred embodiments, the cell viability is selected from 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%.

In another embodiment of the present disclosure, proliferation of MSCs recovered after cryopreservation is measured during 12 weeks post-plating in culture dish. The results depict that proliferation rates are not compromised and moreover, the proliferation rate is shown to be even enhanced. Further, the quality of MSCs post freezing are retained/improved which is depicted by the presence of good number of CFU-F (shown in the below examples section).

The cryopreservation media of the present disclosure is a ready to use cryopreservation media containing low concentration of cryoprotectant wherein there is no need to dilute the concentration of cryoprotectant post thawing of cells or no need of any sort of manipulation to the cells, and the cells can be directly infused or injected to a subject.

In an embodiment of the present disclosure, after thawing the MSCs from the instant cryopreservation media, stability of the product (MSCs) is maintained for 2 to 5 hours at room temperature (21°C to 25 °C).

In another embodiment as described above, high freezing density, excellent cell viability and enhanced proliferation rates of post-cryopreserved MSCs is achieved due to the employment of cryopreservation composition of the instant disclosure.

In yet another embodiment of the present disclosure, the frozen cells are qualitatively and quantitatively analysed for their differential potential wherein MSCs successfully differentiated into adipocytes, osteocytes and chondrocytes.

In a further embodiment of the present disclosure, cells frozen in freezing formulations of the present disclosure retain their differentiation potential. In a still further embodiment of the present disclosure, cells frozen in freezing formulations of the present disclosure show expression of growth factors selected from a group comprising VEGF, TGF-β, PGE-2, ANG-1 and other bio secretory factors known in the art factors, or any combination thereof.

The components of cryopreservation composition of the instant disclosure have a unique role in providing effective cryopreservation of cells. Further, the concentrations of the components of the cryopreservation composition as provided in the instant disclosure are also of utmost importance resulting in enhanced efficacy of said composition in preserving cells. The composition of the present disclosure achieves better cryopreservation and highly viable cells by using low concentrations of cryoprotectant and anti-aging agent. Further, the combination of low concentrations of cryoprotectant, energy substrate and anti-ageing agent increase the efficiency of the freezing composition of the present disclosure.

The composition of the present disclosure can be administered directly as a clinical bedside to a patient in need thereof without any sort of manipulation (such as dilution of DMSO in order to bring down its concentration in the composition) before administration, ultimately rendering the instant composition and cells less toxic and safer to use. The cryopreservation composition of the instant disclosure can be employed for wide range of applications including but not limited to storage for long time-periods, for preparation of cell banks, for transportation of cell products and so on. Further, as described in the above sections and in the forthcoming examples, the frozen cells possess excellent viability, quality/characteristics and stability over long periods.

While the instant disclosure is susceptible to various modifications and alternative forms, specific aspects thereof has been shown by way of examples and drawings and are described in detail below. However, it should be understood that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention as defined by the appended claims. The present disclosure is further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.

EXAMPLES

Materials employed for arriving at the examples of the present disclosure

The materials/reagents employed in the below examples along with sources are provided in Table No. 2.

Table 2: Materials employed

Example 1: Cryopreservation composition and method of preparation

The cryopreservation/freezing composition of the instant disclosure comprises ionic balance buffer, cryoprotectant/cryopreservation antifreeze protectant, protein component, energy substrate and anti-ageing agent. A freezing composition at the below mentioned concentrations of the components are developed and the composition details are as follows:

COMPOSITION 1

Stock solutions of each of the above components (except Plasmalyte A) are made as per manufacturer's instmctions under sterile conditions. Each of the components is then mixed together at the above said v/v percentage and the final volume is made up to 100% by adding Plasmalyte A as recited above in order to obtain the final formulation.

Example 2: Method of freezing/cyropreserving cells using cryopreservation composition

The cells are frozen by subjecting them to composition 1 as described in Example 1. The method of freezing the cells is elaborated below. During culturing, the MSCs are grown from passage 3 (P3) to passage 5 (P5) in one cell stacks in order to get the required number of cells. Once the cell stacks reach confluency, the cells are harvested and frozen in freshly prepared freeze mix/Composition at a concentration of 5 million cells/ml, 10 million cells/ml and 15 million cells/ ml in Nunc® cryovials.

The frozen vials are then transferred to Mr. Frosty Freezing Container (prechilled at 4 degree Celsius) and kept in -80 degree Celsius overnight and subsequently transferred to -196 degree Celsius. The cells of these vials are then tested for their viability by trypan blue- dye exclusion method at different time points namely 0 Week, 2 Week, 4 Week, 8 Week, 12 Week and by flow cytometry using 7AAD marker at 12 week time point. In addition to viability, proliferation kinetics of frozen MSCs were analysed at 2 Week, 4 Week, 8 Week and 12 Week and percentage apoptotic cells are measured using Tali Image based Cytometer. The viability results for these frozen cells are provided in the succeeding examples.

The experiments conducted show that the instant Freezing composition, result in good viability of cells despite containing low concentration of the cryoprotectant. Further, three month (12 weeks) evaluation study show good viability and total cell recovery, proliferation, phenotype expression, multipotent characteristics, and low percentage of apoptic cells. In particular, BM-MSCs frozen in the instant formulation maintain >95% MSCs phenotypic markers expression. Additionally, the cells frozen/cryopreserved in the instant formulation show 85 to 98% viability after repeated freeze-thaw cycles, at different time points. Extensive characterization are carried out at three months (12 weeks) time point, and the morphology of thawed MSCs in the instant formulation is small, fibroblastic, and spindle shaped. Further, proliferation rate is analysed by expanding to one more passage and which shows -44 PDT (population doubling time) with ~5 PD (population doubling). Other potency and functionality tests are also performed to make the cells more viable to use. The viability results for these frozen cells are provided in the succeeding examples.

Example 3: Viability and other studies of MSCs cryopreserved in cyropreservation composition

The mesenchymal stem cells (MSC) are cryopreserved as described in the previous example at concentrations of 5 million, 10 million and 15 million cells per ml of freeze mix in cryovials.

Analysis Time points:

The MSCs are cryopreserved in the freezing formulation at 5 different time points- Time point 1 (week Ό'- in cryopreservation formulation), Time point 2 (week '2'), Time point 3 (week '4'), Time point 4 (week '8'), and Time point 5 (week '12').

Following test methods/endpoint parameters are employed during analysis time points: 1. Viability by Dye Exclusion Method (Trypan Blue) is analysed for all the time points 1 to 5. Further, at time point 5 and proliferation kinetics at time points 2 to 5, extensive characterizations are performed to confirm the quality of frozen cells.

2. Viability by 7- Amino Actinomycin D (7AAD) using flow cytometry.

3. Analysis of viability by Image Based Cytometer (Tal i instrument),

4. Clonogenecity potential (Colony Forming Unit Assay),

5. Qualitative and Quantitative Differentiation (Adipogenic, Osteogenic and Chondrogenic differentiation Potential). 1. Viability by Dye Exclusion Method (Trypan Blue): The frozen MSC's are thawed by carrying out centrifugation. After centrifugation, the cells are resuspended in an appropriate volume of complete media (containing Knockout-DMEM, FBS and Glutamax). The cell suspension is mixed with trypan blue (a test to determine the number of viable and nonviable cells), then applied in the Haemocytometer and visually examined under the microscope for stained and unstained cells. The cells which take up the stain are considered nonviable while the other cells which do not possess the stain are considered as viable cells. The following formula is used to calculate the total number of viable cells and the viability percentage in the cell suspension population. Total viable cells/ml= Total cells counted * dilution factor/No. of squares * 10,000 cells/ml

Here, the dilution factor is the dilution of cell suspension done with trypan blue. For example, 40 μΐ of trypan blue and 10 μΐ of cell suspension is 5 times dilution. The total cells counted are the total number of cells in the 4 corner squares of the Haemocytometer.

Further, the viability % is calculated as follows:

Viability = (Total viable cells /Total cells (viable + nonviable cells) *100.

The results for this assay when the cells were cryopreserved in freezing composition presented in table 3 and in Figure 1 : Table 3: Viability of frozen MSC in freezing composition using Dye Exclusion Method

The above results depict excellent cell viability (as high up to 97%) of MSCs (at cell densities of 5 million, 10 million and 15 million cells/ml when said MSCs are frozen in the cryopreservation composition of the instant disclosure.

2. Viability by 7AAD using flow cytometry: Cell viability was measured by Flow Cytometry using 7- Amino Actinomycin D (7AAD), a chemical which can penetrate through cell membrane of dead cells. 100 μΐ of cell suspension were incubated in the dark for about 20 minutes at R.T with saturating concentrations of Fluorescein Iso-Thiocyanate (FITC) or Phycoerythrin-(PE), conjugated antibodies. Appropriate Isotype matched controls were used to set the instrument parameters. After incubation, cells were re-suspended in about 200 μΐ of Sheath buffer for analysis. Analysis was performed in flow cytometry, BD LSR II (BD Biosciences). Cells were identified by light scatter for 10,000 gated events and analyzed using BD FACS Diva software. The results of this assay are depicted in Figure 2.

The results show that the % viability of cells (week 12) at different freezing densities range from about 98.3 % to about 98.7 % respectively. 3. Analysis of viability by Tali Image based Cytometer: The cells frozen in freezing composition were tested for viability using Tali Image based cytometer and analysis of percentage viability at 3 month (week 12) time point is performed for which the results are provided below: Table 4: Analysis of percentage of apoptotic cells and viability using Tali Image Based

Cytometer

The results of above Table 4 depict that MSCs frozen in the cryopreservation formulation at different cell densities show very low percentage of apoptotic cells at 3 months (12 weeks) time point. In accordance with the above results, it is also observed that cells frozen in the cryopreservation formulation of the instant invention show viability above 95 %. Therefore, cells frozen in composition of the present disclosure retain high viability percentage for a long period of time with a lower rate of apoptosis.

4. Colony Forming Unit (CFU-F) Assay: The MSC's post thawing and centrifugation are resuspended in an appropriate volume of complete media such that the cell concentration remains as 1 million per ml. The cell suspension is serially diluted in order to plate 100 cells per 100 mm dish. Complete media [comprising 10% KO- DMEM (knock out-Dulbecco's modified Eagle's medium) + 2 ng/ml bFGF (basic Fibroblast Growth Factor)] changes are given every 3 ^rd day. The formation of colonies is progressively screened. The staining of the dishes is done on Day 14 from the day of plating the cells.

Figure 5 in conjunction with the below Table 5 depict the results of the above assay i.e. formation of colonies at time point 5 (3 months).

Table No 5: CFU-F assay results

Staining and Counting of colonies: On Day 14, the media is aspirated from the plate and the cells are fixed with 1% Paraformaldehyde for about 20 minutes. IX DPBS wash is given for about 5 minutes. 0.1% Toluidine Blue is added to it and incubated for about 1 hour. The cells are then given 2 consecutive washes for about 5 minutes each with R.O (reverse osmosis) water. The water is aspirated out and the dish is allowed to dry by inverting it on absorbent paper towels and complete air drying. By definition, a colony must have a minimum of 50 cells. The dish is inverted and equal quadrant grids are made on its bottom and the colonies are enumerated. The results of the enumeration of the stem cells (CFU-F efficiency assay) are provided in figure 6B.

The results of 'Figure 6A and 6B' depict that the instant freezing formulation maintains frozen MSC's in an undifferentiated state as good number of CFU-F clones [i.e. presence of Mesenchymal Precursor Cells (MPC)] were found after freeze thaw at 3 month (12 week) time point.

5. Population doubling kinetics: The frozen MSC's at time point 2 to time point 5 of post thawing and centrifugation are re-suspended in an appropriate volume of complete media such that the cell concentration remains 1 Million per ml. The cell suspension is seeded into T75 flasks at the seeding density of 1,000 cells/ cm ² . Complete media [comprising 10% KO- DMEM+ 2 ng/ml bFGF] changes are given every 3 ^rd day. The rate of confluency is progressively screened and the cells are harvested when they are nearly reaching 80-90% confluency. The harvested cell suspension is counted and based on its final harvest count, the population doubling number and time is calculated by the following formula: X = [loglO (NH) - loglO (NI)]/log10(2).

Where NI is the inoculum cell number and NH is the cell harvest number.

The Population doubling time is obtained by the formula:

TD = tplg2/(lgNH - lgNI),

where NI is the inoculum cell number, NH is the cell harvest number, and t is the time of the culture (in hours). The following table 6 provides the results for the population doubling kinetics of the stem cells harvested.

Table 6: Analysis of population doubling kinetics

The results of above Table 6 establish that thawed MSC's show good proliferation potential after seeding to culture dish and the morphology of MSC's frozen in the present composition show small fibroblastic, and spindle shaped cells.

Example 4: Qualitative and Quantitative Differentiation of cryopreserved stem cells

A) Adipogenic differentiation, staining and quantification: The MSC's after thawing and centrifugation are resuspended in an appropriate volume of complete media such that the cell concentration remains 1 Million per ml. The cell suspension is seeded into 6 well plates at the seeding density of 1,000 cells/ cm ² . Complete media [comprising 10% KO- DMEM+ 2 ng/ml bFGF] changes are given every 3rd day. The rate of confluency is progressively screened and the cells are added with Adipogenic differentiation media (StemPro Adipogenic differentiation kit at 9:1 ratio) when they nearly reach 80-90% confluency. The cells are taken forward into staining of adipocytes after 21 days of induction of differentiation. Media changes are given every 3rd day. The cells are washed with IX DPBS after aspiration of differentiation media. The cells are fixed with. 4% Paraformaldehyde and incubated for about 30 minutes at room temperature. The cells are washed twice with DPBS and 0.5% Oil Red O stain is added and incubated for about 1 hour at room temperature followed by about 2 washes with R.O water. The cells are observed under the microscope for oil droplets. Photographs are taken at 20x magnification and the results are depicted in Figure 7A. The Quantification is done spectrometrically on the cells which pick up Oil Red O stain. The cells are added with chloroform: methanol mixture (2:1 ratio) and the eluted stain is read at 540 absorbance in the quartz cuvette measured against a blank solvent using a spectrophotometer (Figure 7B). B) Osteogenic differentiation, staining and quantification: The cryopreserved MSC's after thawing and centrifugation are re-suspended in an appropriate volume of complete media such that the cell concentration remains 1 Million per ml. The cell suspension is seeded into 6 well plates at the seeding density of 1,000 cells/ cm ² . Complete media [comprising 10% KO- DMEM+ 2 ng/ml bFGF] changes are given every 3rd day. The rate of confluency is progressively screened and the cells are added with Osteogenic differentiation media which is obtained from the StemPro Osteogenic differentiation kit when they are at nearly 80-90% confluency. The cells are taken forward into staining of osteoblasts after 21 days of induction of differentiation. Media changes are given every 3rd day. The cells are washed with IX DPBS after aspiration of differentiation media. The cells are fixed with 4% Paraformaldehyde and incubated for about 30 minutes at room temperature. The cells are washed twice with DPBS and 2% Alizarin Red stain added and incubated for about 20 minutes at room temperature. This is followed by 2 washes with R.O water and the cells are observed under the microscope for calcification and osteoblast nodules. Photographs are taken at 10x magnification and the results are depicted in Figure 8.

The morphology of the osteocytes is depicted in figure 8.

C) Chondrogenic differentiation, staining and quantification:

The MSC's after centrifugation are re-suspended in an appropriate volume of complete media such that the cell concentration remains 1 Million per ml. The cell suspension is seeded into 6 well plates at the seeding density of 1,000 cells/ cm ² . Complete 10% KO- DMEM+ 2 ng/ml bFGF media changes are given every 3rd day. The rate of confluency is progressively screened and the cells are added with chondrogenic differentiation media (StemPro Chonrogenic differentiation kit) when they are at nearly 80-90%> confluency. The cells are taken forward into staining of chondrocytes after 21 days of induction of differentiation. Media changes are given every 3rd day.

The cells are washed with IX DPBS after aspiration of differentiation media. The cells are fixed with 4% Paraformaldehyde for about 30 minutes at room temperature. The cells are washed twice with DPBS and 1% Alcian Blue stain is added and incubated for about 1 hour at room temperature followed by 3 washes with 0.1N HCL and 4th wash by R.O water. Thereafter, the cells are observed under the microscope for Chondrogenesis (Figure 9A). Photographs are taken at lOx magnification.

Further, the differentiated cells are stored as a pellet at -80°C for both DNA and sGAG (sulphated glycosaminoglycan) analysis. Sulphated Glycosaminoglycan quantification was carried out using the BlyScan© Sulfated Glycosaminoglycan Qualification Kit after normalizing DNA with Pico green kit. The results in Figure 9B show an increased sGAG expression in the differentiated cells derived from the cells preserved in the instant cryopreservation composition at 3 month (12 weeks) time point.

The results of the above depicted differentiation assays establish that cells frozen in freezing formulations of the present disclosure retain their functional/biological characteristics which is evident from the differentiation of cells frozen in the instant freezing formulation into adipocytes, osteocytes and chondrocytes.

Example 5: VEGF (Vascular Endothelial Growth Factor) Potency Assay

VEGF is a potent angiogenic marker and its potential release by BM- MSCs supports the idea that the paracrine mechanism underpins/attenuates the biological effects of long-term angiogenesis in critical limb ischemia (CLI patients).

Here, the amount of VEGF in the conditioned medium derived from the investigational medicinal product (IMP) [BM-MSCs] - P5 cultures is estimated. The BM-MSCs are cryopreserved/stored in the cryopreservation composition of Example 1 till time point 5 (12 weeks). BM-MSCs are thereafter plated at the density of 1X10 ⁶ cells in a T-75 flask (BD) in duplicates. Conditioned medium is collected at the end of 48 and 72 hours respectively from the T -75 flasks which is fed with DMEM-KO, 10% FBS, Glutamax (100 U/ml), Penstrep (100 U/ml) and 2 ng/ml bFGF. Conditioned medium is also collected from the large scale production batches which are cultured in cell stacks on the day of harvest at 80-90% confluency at Passage 5 (P5). The collected media is spun down at 1500 rpm and filtered with a ().22μ syringe filter (Millipore) stored at -80°C. Human VEGF Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) is used as per the instructions of the manual present in the kit. 200 μΐ of conditioned medium is used for the assay and separate standards are included for each run. The absorbance is read at 450nm using the Spectramax M3 plate reader. The results for this VEGF assay is provided in Figure 10. The results depict that there is an increase in the VEGF marker after 48 hours and 72 hours, thereby establishing the potency of the BM-MSCs for CLI treatment wherein said BM- MSCs are cryopreserved in the cryopreservation composition at different densities for 12 weeks (time point 5).

Example 6: Effect of composition with deviation in the concentration of protein component & cryoprotectant, and lacking anti-ageing agent

A freezing composition (Composition 2) at the below mentioned concentrations of the components [with lack of ectoine and half the amounts of HSA and glycerol vis-a-vis Composition 1] is developed and the composition details are as follows: COMPOSITION 2

Component v/v %

(i) DMSO 5%

(ii) Glycerol 2.5%

(iii) 20% Human Serum Albumin (HSA) 2.5%

(iv) 2.5M Trehalose 0.96%

(v) 2.5M Hydroxyethyl starch 1.47%

(vi) GlutaMax 1%

(vii) Poly L Lysine 0.01%

(viii) PlasmaLyte A 86.56%

The mesenchymal stem cells (MSC) are cryopreserved in Composition 2 following the method as described in Example 2 at concentrations of 5 million (5M) and 10 million (10M) cells per ml of freeze mix in cryovials. Analysis Time points:

The MSCs are cryopreserved in the freezing formulation at 3 different time points- Time point 1 (week 'Ο'- in cryopreservation formulation), Time point 2 (week '2') and Time point 3 (week '4').

The viability of the cryopreserved cells was analysed through Dye Exclusion method (Tryphan blue), during analysis time points 1-3 (Week 0, 2 and 4 respectively).

The effect on Viability of the cryopreserved cells as observed in Dye Exclusion method is as follows:

Table 7: Analysis of cell Viability of frozen MSC in Freezing Composition 2

As observed from results of the Dye Exclusion method (Tryphan blue), reducing the amounts of HSA (protein component) and Glycerol (cryoprotectant), and removing Ectoine (anti- ageing agent) from the present Composition causes reduction in viability of the cryopreserved cells.

It is observed that viability of cells stored in composition 2 is 62.5%> (5M/ml) and 44%> (lOM/ml). This is much lower than the 94.5% (5M/ml and lOM/ml) viability of cells stored in composition 1 of Example 1 at week 0.

At week 2, as observed in Example 3 (1), viability of cells stored in composition 1 at week 2 is 89% (5M/ml and lOM/ml) while that of cells stored in Composition 2 at week 2 is 59% (5 M/ml) and 69% ( 1 OM/ml) respectively.

At the end of week 4 while cells stored in composition 1 show high viability of 91% (5M/ml) and 93% (1 OM/ml) respectively, it can be observed that cryopreservation in Composition 2 reduces viability to 57.44% (5M/ml) and 54.45% (lOM/ml) respectively. In view of the above analysis on cryopreservation efficiency of composition 2, it can be observed that the results are inferior at freezing densities of 5M and 10M cells/ml. Accordingly, it is further understood that if the freezing density is enhanced (for example, up to 15M cells/ml or more), the cell viability would be similar or even inferior than the results achieved at 5M and 10M cells/ml.

Further, it can be observed from the above analysis that reduced viability is observed at week 4 itself when the cells are cryopreserved in composition 2. Accordingly, this inferior result suggests that the viability will gradually decrease if the cells are cryopreserved for a longer period of time.

Therefore, from the above analysis, it can be clearly observed that deviation from the instant Composition (such as composition 1 of Example 1) results in decrease of viability of cryopreserved cells. In other words, although composition 2 having some components might be showing some positive results, it is unable to showcase superior efficiency in cryopreservation as observed when all the components of the present composition (such as composition 1) at the claimed concentrations are employed. This provides for the fact that when all the components viz. ionic buffer, cryoprotectant, protein, energy substrate and anti- aging agents come together, there is synergism which is apparent due to far better results when compared to the results obtained by composition having lesser components along with concentrations falling outside the ranges as provided in the present disclosure.

Example 7; Effect on cryopreservation in a composition lacking protein component and anti-ageing components-L-Glutamine and PoIy-L-lysine

A freezing composition lacking protein component (HSA), L-Glutamine and Poly-L-lysine (Composition 3) at the below mentioned concentrations of the components is developed and the composition details are as follows:

COMPOSITION 3

The mesenchymal stem cells (MSC) are cryopreserved in Composition 3 following the method as described in Example 2 at concentrations of 5 million (5M) and 10 million (10M) cells per ml of freeze mix in cryovials. Analysis Time points:

The MSCs are cryopreserved in the freezing formulation at 3 different time points- Time point 1 (week 'Ο'- in cryopreservation formulation), Time point 2 (week '2') and Time point 3 (week '4'). The viability of the cryopreserved cells was analysed through Dye Exclusion method (Tryphan blue), during analysis time points 1-3 (Week 0, 2 and 4 respectively).

The effect on Viability of the cryopreserved cells as observed in Dye Exclusion method is as follows:

Table 8: Analysis of cell Viability of frozen MSC in Freezing Composition 3

As observed from results of the Dye Exclusion method (Tryphan blue), excluding protein component - HSA, L-Glutamine and Poly-L-lysine from the present Composition causes reduction in viability of the cryopreserved cells.

It is observed that viability of cells stored in composition 3 is 64% (5M/ml) and 55% (lOM/ml). This is lower than the 94.5% (5M/ml and lOM/ml) viability of cells stored in composition of Example 1 at week 0.

At week 2, as observed in Example 3 (1), viability of cells stored in composition 1 at week 2 is 89% (5M/ml and lOM/ml) while that of cells stored in Composition 3 at week 2 is 78% (5M/ml) and 79% (lOM/ml) respectively. At the end of week 4 while cells stored in composition 1 show high viability of 91% (5M/ml) and 93% (10M/ml) respectively, it can be observed that cryopreservation in Composition 3 reduces viability to 68.93% (5M/ml) and 64% (lOM/ml) respectively. In view of the above analysis on cryopreservation efficiency of composition 3, it can be observed that the results are inferior at freezing densities of 5M and 10M cells/ml. Accordingly, it is further understood that if the freezing density is enhanced (for example, up to 15M cells/ml or more), the cell viability would be similar or even inferior than the results achieved at 5M and 10M cells/ml. Further, it can be observed from the above analysis that reduced viability is observed at week 4 itself when the cells are cryopreserved in composition 3. Accordingly, this inferior result suggests that the viability will gradually decrease if the cells are cryopreserved for a longer period of time.

Therefore, from the above analysis it can be clearly observed that deviation from the instant Composition (such as composition 1 of Example 1) results in decrease of viability of cryopreserved cells. In other words, although composition 3 having some components (and lacking components such as protein and anti-ageing agents) might be showing some positive results, it is unable to showcase superior efficiency in cryopreservation as observed when all the components of the present composition (such as composition 1) is employed. This provides for the fact that when all the components viz. ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent come together, there is synergism which is apparent due to far better results when compared to the results obtained by components having lesser components.

Example 8; Effect on cryopreservation with composition lacking energy substrate and ectoine (anti-ageing agent)

A freezing composition lacking energy substrate (trehalose and hydroxyethyl starch) and anti-ageing agent (ectoine) (Composition 4) at the below mentioned concentrations of the components is developed and the composition details are as follows: COMPOSITION 4

The mesenchymal stem cells (MSG) are cryopreserved in Composition 4 following the method as described in Example 2 at concentrations of 5 million and 10 million cells per ml of freeze mix in cryovials.

Analysis Time points:

The MSCs are cryopreserved in the freezing formulation at 2 different time points- Time point 1 (week Ό'- in cryopreservation formulation) and Time point 2 (week T).

The viability of the cryopreserved cells was analysed through Dye Exclusion method (Tryphan blue), for analysis time points (Week 0 and 1)

The effect on Viability of the cryopreserved cells as observed is as follows:

Table 9: Analysis of cell Viability of frozen MSC in Freezing Composition 4

As observed from results of the Dye Exclusion method (Tryphan blue), excluding energy substrate and anti-ageing component - ectoine from the instant Composition (such as composition 1 described in Example 1) causes significant reduction in viability of the cryopreserved cells from week 0 to week 1 itself. This suggests that the viability will further decrease gradually if the cells are cryopreserved for a longer period of time, for instance up to week 4 or above. Further, it can be observed that the results are inferior at freezing densities of 5M and 10M cells/ml of composition 4. Therefore, if the freezing density is further enhanced (for instance, up to 15M cells/ml or more), the cell viability would be similar or even inferior than the results achieved at 5M and 10M cells/ml.

Therefore, from the above analysis, it is clearly observed that deviation from the components of the instant Composition results in decrease of viability of cryopreserved cells. In other words, although composition 4 having some components might be showing some positive results, it is unable to showcase superior efficiency in cryopreservation as observed when all the components of the present composition (such as composition 1) is employed. This provides for the fact that when all the components viz. ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent come together, there is synergism which is apparent due to far better results when compared to the results obtained by components having lesser components (for instance, lack of energy substrate and anti -ageing component - ectoine).

Example 9: Effect of deviation of concentration of anti-ageing agent on cell viability

The cryopreservation/freezing composition of the instant disclosure comprises ionic balance buffer, cryoprotectant/cryopreservation antifreeze protectant, protein component, energy substrate and anti-ageing agent (combination of poly-L-lysine, ectoine and L-Glutamine). A freezing composition comprising increased concentration of anti-ageing component (Composition 5) is developed and the composition details are as follows:

COMPOSITION 5

Table 10: Analysis of Cell Viability of frozen MSC in Freezing Composition 5

As observed from results of the Dye Exclusion method (Tryphan blue), employing higher concentrations of anti-ageing agents compared to the instant Composition 1 (as described in Example 1) causes reduction in viability of the cryopreserved cells. Further, as can be seen in the present example, a higher cell concentration/freezing density was employed when compared to examples 6- 8 and the viability results were still inferior as shown above.

In other words, although composition 5 has all the components viz. ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent, a variation in slightest of amounts (for instance, slight increase in the amount of anti-ageing agent from 2% to 2.2 %) lead to inferior results in cryopreservation when compared to the present composition which contain all the components viz. ionic buffer, cryoprotectant, protein, energy substrate and anti-aging agent within the amounts as specified by the present ranges. Thus, the specific components along with concentration ranges as described in the present disclosure are important to achieve superior cryopreservation results.

Thus, the present disclosure provides an improved and critically evaluated clinical grade cryopreservation composition for cryopreserving cells (particularly stem cells) for prolonged periods at high freezing density. The specific combination of various components and the respective concentrations render the instant cryopreservation composition highly efficient in cryopreserving cells. Further, said composition is a ready to use cryopreservation composition containing low concentration of cryoprotectant, and can be directly infused or injected to a subject in need. The qualitative and quantitative tests on cell viability, immunophenotype, multipotential differentiation capacity and apoptosis have proven that frozen stem cells (MSC's) do not acquire any alteration of any of the above characteristics. Further, enhanced proliferation rates of post-cryopreserved MSCs are observed. In a nutshell, following important conclusions/non-limiting advantages are achieved by the cryopreservation composition of the instant disclosure: i) No cell loss is observed upon thawing the frozen samples at all-time points. Further, post thaw manipulations like removing cryopreservant (such as DMSO), reconstitution, washing and centrifugation, concentration and making pre-filled syringes and so on are not required when the instant cryopreservation composition is employed.

ii) No significant differences in total cell recovery is observed when compared with either low freezing density (5 million cells per ml) or high freezing density (15 million cells per ml) throughout the evaluation period. Further, as high as about 97-100 % of frozen cells are recovered as live cells after freezing in the instant formulation followed by storage in liquid nitrogen for 12 weeks. Additionally, average cell recovery after three months storage is about 85% to about 96%.

iii) Cells cryopreserved with the freezing composition show good viability in spite of containing low concentration of dimethylsulfoxide (DMSO) cryoprotectant (i.e. less than or equal to 5% DMSO) in it. In particular, the compositions show >85% viability after repeated freeze thaw cycles at different time points. Further, this proves that the instant formulation provides for enhanced cell viability and functionality while eliminating the need for proteins and high levels of cytotoxic agents.

iv) MSCs frozen in the composition maintain >95% MSCs phenotypic marker expression.

v) Morphology of MSCs frozen in the instant cryopreservation composition is fibroblastic and spindle shaped.

vi) Freeze thaw MSCs show good proliferation potential after seeding to culture dish. Proliferation of MSG recovered after cryopreservation is measured during 12 weeks post- plating. Proliferation rate is not compromised and is even enhanced.

vii) Good number of CFU-F in the frozen M SCs is observed which proves that the instant cryopreservation composition do not alter the MSC quality.

viii) The frozen cells successfully differentiate into adipo, osteo and chondrocytes proving their stability and retaining multipotency.

ix) MSCs frozen in the instant formulation show low percentage of apoptotic cells at 3 months' time point further strengthening the viability and stability features.

x) No need to remove or dilute the concentration of cryoprotectant (such as DMSO) after thawing the MSCs post preserving in the instant cryopreservation composition. xi) Stability of the cell product post thawing is maintained for more than 2 hours and preferably up to about 5 hours at room temperature (21 to 25 °C).

xii) High cell densities (up to about 25 million cells per ml to about 30 million cells per ml) can be frozen with the instant cryopreservation composition. The same is also evident from the results which demonstrate that stem cells are successfully cryopreserved at high freezing density and can be employed for banking and other clinical applications.

Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided above. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.

The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.