WO2023281524A1

WO2023281524A1 - Placenta derived mesenchymal stromal cell secretome, process and uses thereof

Info

Publication number: WO2023281524A1
Application number: PCT/IN2022/050492
Authority: WO
Inventors: Suma Kantipudi
Original assignee: Suma Kantipudi
Priority date: 2021-07-06
Filing date: 2022-05-26
Publication date: 2023-01-12

Abstract

The present invention provides a GMP compliant, scalable process of a cell-free, xeno-free protein and miRNA rich product derived from human placenta i.e., Mesenchymal stromal cell secretome useful for intradermal/subcutaneous administration for hair regeneration and skin rejuvenation. Also provided is a product obtainable from the process and uses thereof.

Description

PLACENTA DERIVED MESENCHYMAL STROMAL CELL SECRETOME, PROCESS AND USES

THEREOF

FIELD OF INVENTION

The present invention relates to the field of stem cells and regenerative medicine. In particular, the present invention provides secretome derived from human placental Mesenchymal Stromal Cells. The benefits of clinical translation of secretome as a regenerative therapy is also provided.

BACKGROUND OF THE PRESENT INVENTION

Conventional management for skin rejuvenation and hair loss are limited to chemical agents such as finasteride and minoxidil and allogenic platelet rich plasma. However, the side effects due to long-term usage of minoxidil and difficulties in repetitive administration in PRP therapy have restricted the above as standard and approved therapies. There is an unmet need of safe, non toxic proteome derived to suit clinical or cosmeceutical application.

SUMMARY OF THE PRESENT INVENTION

In an aspect of the present invention, there is provided a cell-free conditioned cell culture medium comprising: (a) at least a protein selected from the group consisting of collagen alpha-1

(I), collagen alpha-2, type IV collagenase isoform 2, collagen alpha-1 (III), transforming growth factor-beta induced protein ig-hB, and combinations thereof; and (b) at least a mi-RNA selected from the group consisting of hsa-miR-21-5p, hsa-miR-3191-3p, hsa-miR-100-5p, hsa-miR-let-7i- 5p, hsa-miR-4682-3p, and combinations thereof, wherein, the medium is serum starved cell culture medium, wherein the fold change in at least a protein is 10-95-fold more than in serum free plain media and fold change in at least a mi-RNA is 1.5-4-fold less than in serum fed culture media.

In another aspect of the present invention, there is provided a method of preparing a conditioned cell culture medium, the method comprising: (a) obtaining an enriched population of Mesenchymal Stromal Cells (MSCs); (b) seeding and culturing said enriched population of MSCs; and (c) collecting the cell culture medium, wherein step (a) comprises: (i) contacting at least a tissue with media comprising collagenase type 1 and collagenase type 2, wherein collagenase 1 weight concentration in said media is in the range of 0.04-0.1% and collagenase 2 weight concentration in said media is in the range of 0.04-0.1%; and (ii) isolating enriched fraction of MSCs.

In yet another aspect of the present invention, there is provided a cell-free conditioned cell culture medium obtained by a method of preparing a conditioned cell culture medium, the method comprising: (a) obtaining an enriched population of Mesenchymal Stromal Cells (MSCs);

(b) seeding and culturing said enriched population of MSCs; and (c) collecting the cell culture medium, wherein step (a) comprises: (i) contacting at least a tissue with media comprising collagenase type 1 and collagenase type 2, wherein collagenase 1 weight concentration in said media is in the range of 0.04-0.1% and collagenase 2 weight concentration in said media is in the range of 0.04-0.1%; and (ii) isolating enriched fraction of MSCs.

In still another aspect of the present invention, there is provided a use of cell-free conditioned cell culture medium for use in tissue regeneration, vasculogenesis/angiogenesis, extra cellular matrix regeneration, or hair regeneration, said cell culture medium obtained by a method of preparing a conditioned cell culture medium, the method comprising: (a) obtaining an enriched population of Mesenchymal Stromal Cells (MSCs); (b) seeding and culturing said enriched population of MSCs; and (c) collecting the cell culture medium, wherein step (a) comprises: (i) contacting at least a tissue with media comprising collagenase type 1 and collagenase type 2, wherein collagenase 1 weight concentration in said media is in the range of 0.04-0.1% and collagenase 2 weight concentration in said media is in the range of 0.04-0.1%; and (ii) isolating enriched fraction of MSCs.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Fig.l depicts the results in in-vitro cell migration assay treatment regimen with percent cell migration, in accordance with an embodiment of the present invention.

Fig. 2 depicts the pictorial representation of cell migration of different groups, in accordance with an embodiment of the present invention.

Fig .3 depicts the graphical representation of neovascularization in chorio allantoic membrane assay, in accordance with an embodiment of the present invention. Fig. 4 depicts the pictorial representation of chorio allantoic membrane neovascularization under various test conditions, in accordance with an embodiment of the present invention.

Fig. 5 depicts the graphical representation of animal weight in complete skin excisional wound mice model in different treatment groups, in accordance with an embodiment of the present invention.

Fig. 6 depicts the H&E stain of wound area in complete skin excisional wound mice model in different treatment groups, in accordance with an embodiment of the present invention.

Fig. 7 depicts the depicts the Masson Trichome stain of wound area in complete skin excisional wound mice model in different treatment groups, in accordance with an embodiment of the present invention

Fig. 8 depicts the depicts the Mallory stain of wound area in complete skin excisional wound mice model in different treatment groups, in accordance with an embodiment of the present invention

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a cell-free conditioned cell culture medium comprising: (a) at least a protein selected from the group consisting of collagen alpha-1 (I), collagen alpha-2, type

IV collagenase isoform 2, collagen alpha-l(lll), transforming growth factor-beta induced protein ig-h3, and combinations thereof; and (b) at least a mi-RNA selected from the group consisting of hsa-miR-21-5p, hsa-miR-3191-3p, hsa-miR-100-5p, hsa-miR-let-7i-5p, hsa-miR-4682-3p, and combinations thereof, wherein, the medium is serum starved cell culture medium, wherein the fold change in at least a protein is 10-95-fold more than in serum free plain medium and fold change in at least a mi-RNA is 1.5-3-fold less than in serum fed cell culture medium.

In an embodiment, the fold change in collagen alpha-1 in serum starved cell culture medium is about 93-fold more than in serum free plain medium. In another embodiment, the fold change in collagen apha-2 is serum starved cell culture medium is about 61-fold more than in serum free plain medium. In an embodiment, the fold change in type IV collagenase isoform 2 is about 19- fold more than in serum free plain medium. In another embodiment, the fold change in transforming growth factor beta induced protein ig-h3 is about 12-fold more than in serum free plain medium. In an embodiment, the cell-free conditioned cell culture medium further comprises at least one or more of insulin like growth factor binding protein 7 isoform 2, 72kDa type IV collagenase isoform 1, interstitial collagenase isoform 2, collagen alpha 1(VI) chain isoform 2C2, insulin like growth factor II isoform 1, procollagen C endopeptidase enhancer 1, collagen alpha 2(V) chain, collagen alpha 2(1 V) chain, collagen alpha l(XII) chain long isoform, insulin like growth factor binding protein 4, collagen alpha 1(IV) chain isoform 1, insulin like growth factor binding protein complex acid labile subunit isoform 1, collagen alpha 2(V) chain isoform 1. In an embodiment, the at least one or more protein is up-regulated by about 1.5-10-fold more than in serum fed cell culture medium.

In an embodiment, the cell-free conditioned cell culture medium further comprises at least one or more of insulin like growth factor binding protein 3 isoform b, hepatocyte growth factor like protein isoform 3, insulin like growth factor beta binding protein 2, collagen alpha 5(1 V) chain isoform 1, hepatocyte growth factor activator isoform 1, interleukin enhancer binding factor 2 isoform, insulin like growth factor binding protein 5, and hepatocyte growth factor like protein isoform 2. In an embodiment, the at least one or more protein is down-regulated by about at least 1.1-fold less than in serum fed cell culture medium.

In an embodiment, the cell-free conditioned cell culture medium comprises hsa-miR-21-5p, hsa- miR-3191-3p, hsa-miR-100-5p, hsa-miR-let-7i-5p, hsa-miR-4682-3p. In an embodiment, hsa-miR- 21-5p, hsa-miR-3191-3p, hsa-miR-100-5p, hsa-miR-let-7i-5p, and hsa-miR-4682-3p comprised in the cell-free conditioned cell culture medium are the top 5 represented miRNA species in the medium.

In an embodiment, the fold change in hsa-miR-21-5p miRNA in serum starved cell culture medium is about 1.8-fold less than in serum fed cell culture medium. In an embodiment, the fold change in hsa-miR-3191-3p miRNA in serum starved cell culture medium is about 1.65-fold less than in serum fed cell culture medium. In an embodiment, the fold change in hsa-miR-100-5p miRNA in serum starved cell culture medium is about 2.1-fold less than in serum fed cell culture medium. In an embodiment, the fold change in hsa-let-7i-5p miRNA in serum starved cell culture medium is about 3.6-fold less than in serum fed cell culture medium. In an embodiment, the fold change in hsa-miR-4682-3p miRNA in serum starved cell culture medium is about 2.7-fold less than in serum fed cell culture medium. In an embodiment, the medium is obtained from a mesenchymal stromal cell (MSC) culture. The MSCs are derived from tissue selected from the group consisting of adipose tissue, dental pulp, mobilized peripheral blood, birth derived tissue, and placenta tissue. In a preferred embodiment, the tissue is placenta tissue.

In an embodiment, the medium is obtained is serum starved for 12-20 hours prior to collection. In a preferred embodiment, the media is serum starved for about 16 hours.

The present invention also provides a method of preparing a conditioned cell culture medium, the method comprising: (i) obtaining an enriched population of Mesenchymal Stromal Cells (MSCs); (ii) seeding and culturing said enriched population of MSCs; and (iii) collecting the cell culture medium, wherein step (a) comprises: (a) contacting at least a tissue with media comprising collagenase type 1 and collagenase type 2, wherein collagenase 1 weight concentration in said media is in the range of 0.04-0.1% and collagenase 2 weight concentration in said media is in the range of 0.04-0.1%; and (b) isolating enriched fraction of MSCs.

In an embodiment, said tissue is selected from the group consisting of adipose tissue, dental pulp tissue, mobilized peripheral blood, birth derived tissue, and placenta tissue. In a preferred embodiment, the tissue is placenta tissue.

In an embodiment, step (i) is carried out for 20-40 minutes at 33-39°C, preferably 37°C. In a preferred embodiment, step (i) is devoid of trypsin. In an embodiment, in step (ii) after seeding, MSC confluence is 70-80% by day 20-25 at passage 0. In an embodiment, the MSC purity in the MSC culture of step (ii) is in the range of 90-99%, preferably 98% at passage 3. In an embodiment, in step (ii) the MSC are passaged for up to at least 15 times. In an embodiment, the conditioned cell culture medium is collected from serum starved media, wherein the media is starved of serum for at least 12-20 hours. In a preferred embodiment, the media is serum starved for 16 hours.

The present invention also provides a cell-free conditioned cell culture medium obtained by a process as substantially as described herein. The cell-free conditioned medium is for use in any one or more of tissue regeneration, vasculogenesis/angiogenesis, extra cellular matrix regeneration, or hair regeneration. EXAMPLES

Example 1

Comparison of existing cosmeceutical therapies with the cell-free conditioned cell culture medium of the present invention

Example 2

Advantages of the medium of the present invention

The secretome comprised in the cell free serum starved growth medium converts telogen phase to anagen phase in hair follicle growth cycle; decreases factors like BMP2/4, which are responsible for refractory phase in telogen, thereby converting refractory phase to competent phase in telogen, which induct anagen phase through stem cell activation in the local milieu; activation of signaling pathways like Wnt/ -catenin, which is responsible for anagen induction; regulation of hair follicle growth cycle; activation of resident stem cell milieu by the paracrine molecular signaling for initiation of anagen phase of hair cycle; proteoglycans in the secretome act as natural fillers for stable ECM formation in between aging cells; secretome is rich in angiogenic factors for improving skin vascularity; fibroblast activation leads to fresh collagen deposition for firmer skin.

Example 3

Establishing proof of concept study using animal model with a complete excisional skin wound

A splinted skin excisional wound mice model was made by an impression of diameter 1-cm on depilated dorsum at two regions 1cm apart from each other and thereby the wound is created by using toothed forceps and pointed scissors. The area under the impression is cut with full thickness and injected with the media of the present invention to evaluate skin regeneration, including epidermis, dermis and dermal papilla and hair regeneration.

Example 4

Cell-free xeno-free human placenta derived conditioned medium

The animal component, Fetal Bovine Serum (FBS) is replaced with human umbilical cord fetal serum, which is a newly defined medium described previously (Vibudha Nanduri et.al, Reconstruction of Hyaline Cartilage Deep Layer Properties in 3-Dimensional Cultures of Human Articular Chondrocytes. Orthop J Sports Med. 2014 Jun; 2(6): 2325967114539122).

Example 5

Choice of enzymes for tissue digestion

Trypsin is used routinely for isolation of MSCs from placenta. In previous study using the explant method, it is suggested that degradation of the extracellular matrix and disintegration of cell membranes may cause cellular damage through the use of trypsin alone of a long period. In the present case, the combination of collagenase type I and type II are used to digest the tissue, to the exclusion of trypsin, which results in unexpected and surprising better cell release. Enzymatic digestion by collagenase type I and II was found to be more efficient for cell isolation and culturing compared to other known methods described in the art for MSC derivation. Use of collagenase type I and II resulted in shorter time taken to reach 70-80% confluency compared to known methods. Compared to implant technique, using the present process, less contamination and faster growth of cells were seen in the present cell suspension method. Collagenase type I and type II mediated enzymatic digestion of tissue had better efficiency of cell release that trypsin and collagenase type I or trypsin and collagenase type II. Cells were 90% confluent after 9-12 days compared to 14-18 days in conventional methods.

Example 6

Synergistic effect of collagenase type I and type II in placental MSC isolation and expansion

Placental tissue was digested with collagenase type I or collagenase type II individually with trypsin. In each case, it was observed that dissociation of tissue was slow. In contrast, the application of both collagenase type I and II unexpectedly and surprisingly, led to faster tissue dissociation. The placental MSCs derived by this method also reached 70-80% confluency in a shorter time period of 20-25 days at passage 0.

Example 7

Use of human umbilical cord (UC) serum for xeno free cell culture conditions and as inhibitor of collagenase

The enzymatic degradation by collagenases is essential for cell derivation process as described previously to maximize cell yield. There are several inhibitors of collagenases such as EDTA (Ethylenediaminetetraacetic acid), EGTA (ethylene glycol-bis( -aminoethyl ether)-N,N,N',N'- tetra acetic acid),a2-macroglobulin, Cysteine, histidine, DTT (Dithiothreitol), 2-mercaptoethanol, O-phenanthroline, metallic ions such as Hg²⁺, Pb²⁺, Cd²⁺, Cu²⁺. However, human UC serum has a2- macroglobulin as a major component, which can inhibit enzyme activity of collagenases to prevent cellular damage. Advantageously human UC serum can be used for xeno-free cell culture method for safe use in therapeutic applications. Further, growth factor enriched serum also makes it an efficient supplement for cells.

Example 8

Cell derivation process and media collection Placenta, umbilical cord and amniotic membrane are collected after LSCS, washed with saline for

10-15 minutes to remove blood and then suspended in lx PBS until the tissue become pale pink in colour. Next, the tissue is cut into small fragments followed by mincing in a stirrer liquidizer for 15-25 seconds (10000RPM) followed by enzymatic digestion for 30-45 minutes at 37°C. The enzymatic digestion is done with collagenase type I (60mg) and type II (60mg) reconstituted in 30ml PBS. The enzymatic reaction is neutralized with 10% human umbilical cord serum with

DMEM and filtered using 250-micron sieve. It is then centrifuged at 1500RPM for 20 minutes and the pellet is dissolved in DMEM with human umbilical cord serum and plated in T75 vented flasks. The medium is changed every 3-4 days until the cells reach confluency of about 70-80% in about 5-7 days, post which, the supernatant is collected (supernatant collected is post serum starvation). The supernatant collected is centrifuged at 500g for 10 minutes and filtered using

0.2-micron filter and stored for further use.

Example 9 in-vitro cell migration assay/wound healing assay

To determine the wound healing propensity of HeLa cancer cells in presence of the collected supernatant/media, the cells were first seeded onto a 6 well plate. The cells were incubated until they reached full confluence (>95%). Using a sterile lOul tip, a scratch was introduced onto each well of 6 well plate. The wells were washed thrice with plain medium and incubated in presence of supernatant with different concentrations mixed with media. The cells were incubated for 24h at 37°C in CO2 incubator. After the treatment, the cells were washed twice with Dulbecco's Phosphate Buffered Saline and the images were taken in inverted microscope. The scratch distance before and after the treatment was recorded as using image J software and the graph was plotted. The experiment was done in triplicate and the statistical significance (one way ANOVA) was calculated.

Figure 1 depicts the graphical representation of wound healing assay using escalating doses of secretome and compared with control (untreated group)

As seen in Fig. 2 escalating doses of secretome clearly inhibits cell migration in tumor cells (Hela cell line) which gives a direction into therapeutic dose determination in regenerative medicine in preventing tumorigenicity.

At higher doses of secretome, inhibition of migration of cancer cells (Hela cells) is observed, which indicates that the media derived from a fetal tissue exhibits a balance between canonical Wnt signaling and sonic hedgehog pathway thereby balancing over expression of Wnt signaling pathway which prevents migration and proliferation of abnormal cells. Example 10

A comparative study of chick embryo chorio allantoic membrane assay to check the neovascularization following hMSC derived secretome injection:

Fertilized 8 days old chick embryos were incubated at 37° C under 85% humidity. 11-day old egg were used for the experiment. The eggs were sterilized using surgical spirit and the blood vessels was marked using the Egg Candler 1 day prior to inoculation. A hole was made carefully using a sterile needle along the side of the egg. 200mI of supernatant was inoculated and sealed. 10⁶ cells were inoculated onto the CAM layer through this hole as a positive control and later sealed using wax. These cells were further inoculated for 3 days in the incubator. The cells were harvested by break opening the egg shell and separating the CAM layer. The CAM layer was placed onto the petri dish and the neovascularization was quantified by counting the number of blood vessel branch points. A graph was plotted with treatment on x-axis and blood vessels formed on y-axis.

Fig. 3 depicts the histogram representing the neovascularization effect of secretome in the in- ovo CAM assay

Addition of the media/secretome of the present invention appeared to improve wound healing at low concentrations when compared to untreated cells. The secretome appeared to have angiogenic effect on the chorio allantoic membrane of chick embryo, which can be observed from the number of capillaries and their branching. These effects are mediated by their incorporation into newly formed blood vessels or paracrine secretion of proangiogenic factors that stimulate the host chicken endothelial cells. The angiogenic effects of secretome were comparable with the positive control i.e., Hela cells.

As seen in Fig. 4, chorio allantoic Membrane to test the angiogenesis. 5 treatments to test the angiogenic potential of the secretome. a) placebo, b) secretome, c) HeLa (positive control), d) Hela + SeNP and e) secretome+ SeNP (Selenium nanoparticle: negative control) were performed

Example 11

Toxicity study

Three Male Balb C mice around 10 weeks weighing about 25-30 grams was used in Primary Irritation Test. Animals were restrained by holding the tail and applying depilatory cream on their back and the cream was wiped off with sterile cotton after 2 minutes. The area was then sterilized with 70% isopropanol. 2 patches (10 units in each patch; 1cm apart) of secretome derived from human placental MSCs was injected subcutaneously to all the mice and observed after 5 min, 15 min, 30 min and 60 minutes and after 24 hours, 48 hours and 72 hours. Observations for skin inflammation, erythema at the site of injection were performed at least once in a day for 3 days. Also observed were skin sensitizing properties like changes in eye, skin, mucous membrane; respiratory functions, behavioural patterns, body weight, feed intake, water consumption. Special attention was paid at the site of injection. No hypersensitivity reaction was observed in all the 3 mice, with no changes in body weight, feed intake and activity of the animal. No changes were observed at the site of injection. No significant behavioural patterns like seizures, convulsions were observed. No changes in respiratory functions were observed. Example 12

Animal study in regeneration of skin (epidermal, dermal, dermal papilla) and hair

A total of 26 (6+14+6) BALB-C strain mice of male sex (age 8 weeks, weight 25-35g) were grouped into Group-1 (PBS) containing 6 mice, Group-2 (Secretome) containing 14 mice and Group-3 (PRP) containing 6 mice. Animals were under pellet feeding and provided with ad libitum water throughout experiment. Animals were housed in cages following caging standards of mice, each animal per one cage. Animals were handled under care throughout the experiment and humane practices followed for euthanizing the animals. Animals euthanized with high dosage of anesthesia intraperitoneally.

Preparation of excision wound model in mice After anesthetization, about 0.25ml blood was collected from 3 animals randomly from retro- orbital sinus. Dorsum of the animal is depilated and rubbed with 70% ethyl alcohol. An excisional wound was made by an impression of diameter 10mm on depilated dorsum at two regions 1cm apart from each. Wound was created by using toothed forceps and pointed scissors the area under impression was cut with full thickness. After excision of wound, 100 microliters of PBS administered to Group 1 (control) animals, 100 microliters secretome administered for Group-2 and 100 microliters of PRP administered to group 3 (positive control), 50 microliters at each wound given at the center and around the wound at all clock positions subcutaneously. The silicone splint of diameter 10mm placed on the excised wounds and sutured at three points on wound to keep the splints intact. Each animal was caged separately and observed for 14 days. Daily imaging of the animals, wound re-epithelialization and contraction was observed, weights of animals was recorded on all days. Digital quantification of wound was evaluated on day 0, day 1, day 7, day 10 and dayl4.

On day 10, from each group of 3 animals, total 9 animals were sacrificed by high dosage of anesthesia. About 1ml blood was collected through retro-orbital sinus from each animal in EDTA

IB coated tubes and clot activator for serum separation. The wound area was cut along with the normal skin from each wound and kept in 10% formalin. Further these tissues were used for H&E staining, collagen staining and for immunohistochemistry procedures. Same procedure was followed on day 14 to extract the tissue for staining and immunohistochemistry. Fig. 5 depicts the graphical representation of animal weights in complete skin excisional would mice model in different treatment groups. Changes in body weight of animals in different treatment groups. The animals in different treatment groups gained weight throughout the period of study. However, there is a decline in the animal weights in PBS, PRP group when compared with secretome group. Fig. 6 depicts the H&E stain images. Fig. 6A shows PBS group Day 10; minimal cell proliferation, granuloma formation and fibrosis is evidenced, irregular epithelialization, disorientation of collagen matrix is observed; Fig. 6B shows PBS group Day- 14; Thick layer of epithelialization, collagen formation is decreased comparatively with the day 10, increased granulation; Fig. 6C shows hMSC-Secretome group Day-10; Systematic collagen organization & cellular proliferation & migration is noticed; Fig. 6D shows hMSC-Secretome group Day-10; Neovascularization is observed; Fig. 6E shows hMSC-secretome group day-14; new collagen deposition in the newly formed ECM noted; Fig. 6F shows PRP group Day-10; Scab formation is seen, disorientation of the Extracellular matrix, vascularization is noticed; Fig. 6G shows PRP group Day-14; cellular proliferation becoming less compared to day 10, no ECM deposition no cell migration is seen supporting EMT (Epithelial Mesenchymal Transition), complete absence of de novo regeneration of hair follicle.

H & E staining of skin tissues demonstrates inflammatory cells infiltration and found to be low in secretome group when compared with PRP group (positive control) and PBS group (untreated, negative control). The severe inflammatory cell infiltration, very minimal epithelial cell proliferation and granuloma formation was observed in PBS group. In PRP group, there was mild inflammatory cell infiltration along with epithelial cell proliferation (on day 10 but decreased by day 14) with no epithelial cell migration to support EMT. In secretome group, we have observed very mild inflammatory cell infiltration, epithelial cell proliferation and migration (Epithelial- Mesenchymal Transition). There is a complete restoration of all the layers of the skin epidermis and the dermis. The activation of EMT is clearly noted in the H & E with the epidermal cells migrating into the dermis and also activating the mesenchyme to form the dermal papilla.

Fig. 7 depicts Masson Trichome stain images. Fig. 7A shows PBS group Day 10; No cellular differentiation, absence of dermal papilla, severe fibrosis and migration of fibrous tissue; Fig. 7B shows PBS group Day- 14; No de novo hair follicles seen, vascularization not noticed; Fig. 7C shows hMSC-Secretome group Day-10; Cell proliferation & migration with the deposition of new collagen in the ECM; Fig. 7D shows hMSC-secretome group day-14; Structural remodeling of the full thickness skin with new collagen deposition; Fig. 7E shows hMSC-secretome group day-14; De novo Dermal papilla regeneration with complete skin modelling; Fig. 7Fshows PRP group Day- 10; Partial re-epithelialization, severe fibrosis migration of fibrosis; Fig. 7G shows PRP group Day-

14; Absence of hair follicles in the wounded area, decreased vascularization, decreased EMT; Fig. 7H shows PRP group Day-14; No cellular migration and proliferation, instead noticed extension of the fibrosis into the deeper layers.

Masson trichome stain is a marker to assess granuloma and extracellular matrix formation analyzed through collagen fiber deposition. In our experiment, we have found in secretome group, that the collagen deposition and their orientation in ECM was not deviated from the normal skin indicating that the collagen deposition due to secretome would not results in fibrotic scar formation.

Fig. 8 shows Mallory stain images for hMSC secretome group. Fig. 8A shows hMSC secretome group day-10; Complete Epidermal regeneration and migration (EMT) into the dermis with a de novo hair follicle regeneration; Fig. 8B shows hMSC secretome group day-10; Complete re- epithelialization, neovascularization noticed; Fig. 8C shows hMSC Secretome group day- 14; De novo hair follicle regeneration is seen, collagen formation is noticed; Fig. 8D shows hMSC Secretome group day-14; Neovascularization is noticed; Fig. 8E shows hMSC Secretome group day-14; Keratin formation, epidermal proliferation and epidermal cell migration into the dermis

(mesoderm) with the dermal papilla formation, sub-epidermal thin collagen deposition and structural remodeling of the entire skin and hair follicle.

Mallory stain of skin tissue will be applied in detecting the collagen and iron depositions (blood vessels). The secretome group demonstrated that there were collagen fibers and bundles organized in a more regular fashion when compared to PBS or PRP group with marked induction of neovascularization in the wound area.

Overall, the present invention provides a cell-free conditioned MSC culture medium, which has unique functionality in skin and hair regeneration. Also, particularly, a novel and inventive method of MSC culturing is provided which is superior than known methods in the art.

Claims

I/We claim:

1. A cell-free conditioned cell culture medium comprising: a. at least a protein selected from the group consisting of collagen alpha-l(l), collagen alpha-2, type IV collagenase isoform 2, collagen alpha-l(lll), transforming growth factor-beta induced protein ig-hB, and combinations thereof; b. at least a mi-RNA selected from the group consisting of hsa-miR-21-5p, hsa-miR- 3191-3p, hsa-miR-100-5p, hsa-miR-let-7i-5p, hsa-miR-4682-3p, and combinations thereof, wherein the medium is serum starved cell culture medium, wherein the fold change in at least a protein is 10-95-fold more than in serum free plain medium and fold change in at least a mi-RNA is 1.5-4-fold less than in serum fed cell culture medium.

2. The medium as claimed in claim 1, wherein said medium is obtained from a mesenchymal stromal cell (MSC) culture.

3. The medium as claimed in claim 2, wherein the MSC is derived from tissue selected from the group consisting of adipose tissue, dental pulp, mobilized peripheral blood, birth derived tissue, and placenta tissue, preferably placenta tissue.

4. The medium as claimed in claim 1, wherein the cell media is serum starved for 12-20 hours, preferably 16 hours.

5. The medium as claimed in claim 1, wherein the medium is obtained by a method as claimed in claim 6.

6. A method of preparing a conditioned cell culture medium, the method comprising: (i) obtaining an enriched population of Mesenchymal Stromal Cells (MSCs); (ii) seeding and culturing said enriched population of MSCs; and (iii) collecting the cell culture medium, wherein step (i) comprises: a. contacting at least a tissue with media comprising collagenase type 1 and collagenase type 2, wherein collagenase 1 weight concentration in said media is in the range of 0.04-0.1% and collagenase 2 weight concentration in said media is in the range of 0.04-0.1%; and b. isolating enriched fraction of MSCs.

7. The method as claimed in claim 6, wherein said tissue is selected from the group consisting of adipose tissue, dental pulp tissue, mobilized peripheral blood, birth derived tissue, and placenta tissue, preferably placenta tissue.

8. The method as claimed in claim 6, wherein step (ia) is carried out for 20-40 minutes at 33- 39°C, preferably 37°C.

9. The method as claimed in claim 6, wherein step (ia) is devoid of trypsin.

10. The method as claimed in claim 6, wherein in step (ii) after seeding, MSC confluence of 70-80% is achieved in 20-25 days at passage 0.

11. The method as claimed in claim 6, wherein said MSC purity in the MSC culture of step (ii) is in the range of 90-99%, preferably 98% at passage 3.

12. The method as claimed in claim 6, wherein in step (ii) the MSC are passaged for up to at least 15 times.

13. The method as claimed in claim 6, wherein the conditioned cell culture medium is collected from serum starved media, wherein the media is starved of serum for at least 12-20 hours, preferably 16 hours.

14. Cell-free conditioned cell culture medium obtained by a process as claimed in claim 6.

15. Use of cell-free conditioned cell culture medium as claimed in claim 14 for use in tissue regeneration, vasculogenesis/angiogenesis, extra cellular matrix regeneration, or hair regeneration.