Production method of anti-aging mesenchymal stem cells

Abstract

The invention discloses a production method of mesenchymal stem cells applied to anti-aging, and relates to the technical field of cell biologication.The production method of the mesenchymal stem cells applied to anti-aging comprises the following steps that S1, cell culture is conducted, specifically, mesenchymal stem cells are cultured in vitro till the cell fusion degree reaches 60%-95%; s2, cytokine stimulation: adding cytokines into the cultured cell culture medium, and stimulating for 24-48 hours; s3, detecting and screening: digesting and collecting the mesenchymal stem cells, detecting the expression level of indoleamine 2, 3-dioxide oxidase and related cytokines in the mesenchymal stem cells, and detecting the immune regulation ability of T cells; the mesenchymal stem cells obtained by adopting the production method of the mesenchymal stem cells applied to anti-aging provided by the invention have a better anti-aging effect, the immune regulation ability and the specific factor secretion level of the mesenchymal stem cells are improved by adding cytokines, the production method of the mesenchymal stem cells is established, and the mesenchymal stem cells can be used for preparing the anti-aging mesenchymal stem cells. The pharmaceutical composition is applied to anti-aging related diseases.

Description

Technical Field

[0001] The present invention relates to the technology of cell biology field, and in particular to a method for producing mesenchymal stem cells used for anti-aging. Background Art

[0002] Human aging is a complex biological process involving the gradual degeneration of cells, tissues, and organs. This process is usually accompanied by a variety of physiological and functional changes, which ultimately affect the health and quality of life of individuals. Biomarkers of aging include shortening of cell telomeres, increased levels of oxidative stress, and elevated inflammatory markers; in addition, factors released by the senescence-associated secretory phenotype (SASP) can promote chronic inflammation and induce normal cells to enter a senescent state. Inflammation is considered to be an endogenous factor of aging, so eliminating inflammation may become a potential strategy for anti-aging.

[0003] Mesenchymal Stem Cells (MSC) are a type of adult stem cells that are derived from perinatal tissues such as the umbilical cord, umbilical cord blood, and placenta, as well as adult tissues such as bone marrow, fat, and teeth. Due to their multidirectional differentiation potential and immunomodulatory function, mesenchymal stem cells have attracted much attention in their application in diseases such as knee osteoarthritis, inflammatory bowel disease, systemic lupus erythematosus, diabetes, and lower limb ischemia. At the same time, mesenchymal stem cells can promote tissue repair and regulate inflammatory responses by secreting a variety of cytokines (such as EGF, VEGF, Ang-1, FGF, HGF, NGF, SOD, IDO, TGF-β, etc.). Therefore, mesenchymal stem cells are also regarded as an effective means of applying to anti-aging-related diseases, and corresponding clinical data have been accumulated in the field of anti-aging.

[0004] Although mesenchymal stem cells have shown potential in anti-aging applications, the number of cytokines they conventionally secrete is limited, which results in unstable effects and a short duration of therapeutic effects when used to treat aging-related diseases. In order to solve these problems and improve the anti-aging effects of mesenchymal stem cells, the present invention proposes a method for producing mesenchymal stem cells for anti-aging applications, by stimulating mesenchymal stem cells with specific cytokines to enhance their cytokine secretion, thereby achieving a more significant anti-aging effect. Summary of the invention

[0005] In view of this, the present invention aims to address the deficiencies in the prior art, and its main purpose is to provide a method for producing mesenchymal stem cells for anti-aging. The mesenchymal stem cells obtained by adopting the method for producing mesenchymal stem cells for anti-aging provided by the present invention have better anti-aging effects. By adding cytokines, the immune regulation ability of mesenchymal stem cells and the secretion level of specific factors are improved, thereby establishing a method for producing mesenchymal stem cells for anti-aging related diseases.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions: A method for producing mesenchymal stem cells for anti-aging, comprising the following steps: S1. Cell culture: Mesenchymal stem cells were cultured in vitro until the cell confluence reached 60%-95%; S2, cytokine stimulation: add cytokines to the culture medium of the cells and stimulate for 24h-48h; S3. Detection and screening: digest and collect mesenchymal stem cells, detect the expression levels of indoleamine 2,3-dioxygenase (IDO) and related cytokines in mesenchymal stem cells, and detect the immune regulation ability of T cells. Increase the IDO expression level by more than 1,000 times to select mesenchymal stem cells for anti-aging applications.

[0007] As a preferred solution, the in vitro culture of mesenchymal stem cells in step S1 is specifically as follows: the mesenchymal stem cells are cultured at a density of 5000-6000 cells / cm 2 The cells were inoculated at a high density and cultured in αMEM medium supplemented with 5% human platelet lysate at 37°C in a 5.0% CO2 incubator.

[0008] As a preferred embodiment: the concentration of the cytokine in step S2 is 10-50 ng / mL, and the cytokine includes one or a combination of IFN-γ, TNF-α, IL-17A or IL-1β.

[0009] As a preferred solution, the digestion and collection of mesenchymal stem cells in step S3 is specifically as follows: first remove the culture supernatant, rinse the bottom of the culture container with PBS and then remove it, add trypsin to digest the cells for 3 minutes, and then add 2 times the volume of culture medium to terminate the digestion process; take out the cell suspension, centrifuge at 300g for 10 minutes, remove the supernatant, and collect the mesenchymal stem cells.

[0010] As a preferred solution: in step S3, the expression levels of indoleamine 2,3-dioxygenase (IDO) and related cytokines in mesenchymal stem cells are detected by qPCR method, and the related cytokines include ANG-1, NGF, HGF and SOD.

[0011] As a preferred solution: the qPCR method includes: RNA extraction: using TRIzol reagent to extract total RNA from mesenchymal stem cells; cDNA synthesis: using a reverse transcription kit to reverse transcribe the extracted RNA into cDNA; qPCR reaction: designing specific primers for IDO, ANG-1, NGF, HGF and SOD; preparing a qPCR reaction system: adding cDNA samples and performing real-time fluorescence quantitative PCR reaction; data analysis: calculating the relative expression level by the 2^(-ΔΔCt) method, and using GAPDH as an internal reference gene for standardization.

[0012] As a preferred solution: the T cell immune regulation ability is detected in step S3 by a co-culture method, which includes: labeling peripheral blood mononuclear cells (PBMCs); then stimulating CFSE-labeled PBMCs with 10 ng / ml phytohemagglutinin, and co-culturing with umbilical cord mesenchymal stem cells at a ratio of 5:1 at 37°C and 5% CO2 for 4-5 days.

[0013] As a preferred embodiment, the peripheral blood mononuclear cells (PBMCs) are labeled with carboxyfluorofluorescein ethyl succinimidyl ester (CFSE).

[0014] As a preferred solution: after the co-culture is completed for 4-5 days, all lymphocytes are collected and CFSE fluorescence is detected by using a flow analyzer.

[0015] As a preferred embodiment: the mesenchymal stem cells in step S1 are derived from umbilical cord tissue, umbilical cord blood tissue, placental tissue, adipose tissue, bone marrow tissue or dental pulp tissue.

[0016] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical scheme that the mesenchymal stem cells obtained by adopting the mesenchymal stem cell production method for anti-aging provided by the present invention have better anti-aging effects. By simply adding cytokines, the immune regulation ability of mesenchymal stem cells and the secretion level of specific factors are improved, and a method for producing mesenchymal stem cells is established, which is applied to anti-aging related diseases; the method is highly operational and has significant effects; the enhanced anti-aging effect includes improving the low inflammatory state in the body caused by aging or obesity, thereby reducing the risks of related diseases such as diabetes and increased blood lipids.

[0017] In order to more clearly illustrate the structural features and effects of the present invention, it is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram showing the comparison of IDO expression in mesenchymal stem cells of the embodiment of the present invention and the comparative example; Figure 2 A schematic diagram showing the comparison of the expression of the anti-aging-related cytokine ANG-1 in mesenchymal stem cells of the embodiment of the present invention and the comparative example; Figure 3 A schematic diagram showing the comparison of the expression of the anti-aging-related cytokine NGF in mesenchymal stem cells of the embodiment of the present invention and the comparative example; Figure 4 A schematic diagram showing the comparison of the expression of the anti-aging-related cytokine HGF in mesenchymal stem cells of the embodiment of the present invention and the comparative example; Figure 5 A schematic diagram showing the comparison of the expression of the anti-aging-related cytokine SOD in mesenchymal stem cells of the embodiment of the present invention and the comparative example; Figure 6 A schematic diagram comparing the immunomodulatory ability of mesenchymal stem cells in the embodiment of the present invention and the comparative example; Figure 7 A schematic diagram showing the comparison of SOD expression of the anti-aging effect of mesenchymal stem cells in the embodiment of the present invention and the comparative example; Figure 8 A schematic diagram showing the comparison of MDA expression of the anti-aging effects of mesenchymal stem cells in the embodiment of the present invention and the comparative example; Fig. 9 Schematic diagram showing the comparison of IL-10 expression between the anti-aging effects of mesenchymal stem cells of the embodiment of the present invention and the comparative example. DETAILED DESCRIPTION

[0019] The present invention Figure 1 As shown in FIG9 , a method for producing mesenchymal stem cells for anti-aging comprises the following steps: S1. Cell culture: Mesenchymal stem cells were cultured in vitro until the cell confluence reached 60%-95%; S2, cytokine stimulation: add cytokines to the culture medium of the cells and stimulate for 24h-48h; S3. Detection and screening: digest and collect mesenchymal stem cells, detect the expression levels of indoleamine 2,3-dioxygenase (IDO) and related cytokines in mesenchymal stem cells, and detect the immune regulation ability of T cells. Increase the IDO expression level by more than 1,000 times to select mesenchymal stem cells for anti-aging applications.

[0020] The in vitro culturing of mesenchymal stem cells in step S1 is specifically as follows: the mesenchymal stem cells are cultured at a density of 5000-6000 cells / cm 2 The cells were inoculated at a high density and cultured in αMEM medium supplemented with 5% human platelet lysate at 37°C in a 5.0% CO2 incubator.

[0021] The concentration of the cytokine in step S2 is 10-50 ng / mL, and the cytokine includes one or a combination of IFN-γ, TNF-α, IL-17A or IL-1β.

[0022] The digestion and collection of mesenchymal stem cells in step S3 are specifically as follows: first remove the culture supernatant, rinse the bottom of the culture container with PBS and then remove it, add trypsin to digest the cells for 3 minutes, and then add 2 times the volume of culture medium to terminate the digestion process; take out the cell suspension, centrifuge at 300g for 10 minutes, remove the supernatant, and collect the mesenchymal stem cells.

[0023] In step S3, the expression levels of indoleamine 2,3-dioxygenase (IDO) and related cytokines in mesenchymal stem cells are detected by qPCR method, and the related cytokines include ANG-1, NGF, HGF and SOD.

[0024] The qPCR method includes: RNA extraction: using TRIzol reagent to extract total RNA from mesenchymal stem cells; cDNA synthesis: using a reverse transcription kit to reverse transcribe the extracted RNA into cDNA; qPCR reaction: designing specific primers for IDO, ANG-1, NGF, HGF and SOD; preparing the qPCR reaction system: adding cDNA samples and performing real-time fluorescence quantitative PCR reaction; data analysis: calculating the relative expression level by the 2^(-ΔΔCt) method, and using GAPDH as the internal reference gene for standardization.

[0025] In step S3, the T cell immune regulation ability is detected by a co-culture method, which includes: labeling peripheral blood mononuclear cells (PBMCs); then stimulating the CFSE-labeled PBMCs with 10 ng / ml phytohemagglutinin, and co-culturing with umbilical cord mesenchymal stem cells at a ratio of 5:1 at 37°C and 5% CO2 for 4-5 days.

[0026] Peripheral blood mononuclear cells (PBMCs) were labeled with carboxyfluorofluorescein ethyl succinimidyl ester (CFSE).

[0027] After the co-culture for 4-5 days, all lymphocytes were collected and CFSE fluorescence was detected by using a flow cytometer.

[0028] In step S1, the mesenchymal stem cells are derived from umbilical cord tissue, umbilical cord blood tissue, placental tissue, adipose tissue, bone marrow tissue or dental pulp tissue.

[0029] Example: A method for producing mesenchymal stem cells for anti-aging S1. Cell culture: Resuscitation of P2 umbilical cord mesenchymal stem cells from three different donors from the cell bank. The resuscitation method is as follows: open the water bath and adjust the temperature to 37°C; place the cells in the water bath and quickly rewarm them for 3 minutes; when all the cells are dissolved, the resuscitation is complete; After the cells were recovered, they were taken out and mixed with stem cell culture medium (αMEM medium + 5% human platelet lysate) and inoculated in 75cm 2 In the culture bottle, the inoculation density is 5000-6000 cells / cm 2 , cultured for 3-5 days at 37°C, 5.0% CO2, and harvested cells when the confluence reached 85-95%, which were P3 cells; P3 cells were mixed with stem cell culture medium and seeded again in 75 cm 2 In the culture bottle, the inoculation density is 5000-6000 cells / cm 2 , cultured for 3-5 days at 37°C, 5.0% CO2, and harvested cells when the confluence reached 85-95%, which were P4 cells; S2. Cytokine stimulation: P4 cells were mixed with stem cell culture medium and seeded again in 75 cm 2 In the culture bottle, the inoculation density is 5000-6000 cells / cm 2 The culture conditions were 37°C and 5.0% CO2. The cells were cultured until the confluence reached 70%. All the culture supernatant was removed and IFN-γ cytokine was added at a concentration of 15 ng / mL. The culture supernatant was transferred to the culture bottle again and cultured for 24 h. S3. Detection and screening: After the time is reached, the culture supernatant is removed, the bottom of the culture container is rinsed with PBS and then removed, trypsin is added to digest the cells for 3 minutes, and then 2 volumes of culture medium are added to terminate the digestion process; the cell suspension is taken out, and trypsin is added to the culture bottle to digest and harvest the P5 generation cells. The cell suspension is centrifuged at 300g centrifugal force for 10 minutes, the supernatant is removed, and the cell pellet is resuspended in a freezing solution containing 5% DMSO, cooled using a program cooling device, and stored in a liquid nitrogen tank; After 3 days, the frozen cells were taken out and revived again to detect the expression levels of IDO, ANG-1, NGF, HGF, and SOD, and the ability to regulate T cell immunity; The expression levels of IDO, ANG-1, NGF, HGF, and SOD were measured by qPCR, including: 1. RNA extraction: total RNA was extracted from cells using TRIzol reagent; 2. cDNA synthesis: the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit. 3. qPCR reaction: specific primers were designed for IDO, ANG-1, NGF, HGF, and SOD; 3. qPCR reaction system was prepared, cDNA samples were added, and real-time fluorescence quantitative PCR reaction was performed; 4. Data analysis: relative expression was calculated by the 2^(-ΔΔCt) method, and GAPDH was used as the internal reference gene for standardization.

[0030] The co-culture method was used to detect the immunoregulatory ability of T cells: the method included taking peripheral blood mononuclear cells (PBMCs) and labeling them with carboxyfluorescein ethyl ester succinimidyl ester (CFSE). After labeling, 10ng / ml phytohemagglutinin was used to stimulate CFSE-labeled PBMCs, and co-cultured with umbilical cord mesenchymal stem cells at a ratio of 5:1 at 37°C and 5% CO2 for 4-5 days; after the culture, all lymphocytes were collected, and the CFSE fluorescence was detected by flow cytometry to determine the cell proliferation. PBMCs that were not co-cultured were used as a negative control group. The results were expressed as the inhibition rate of human umbilical cord mesenchymal stem cells on PBMCs, inhibition rate = (control group PBMCs proliferation rate - experimental group PBMCs proliferation rate) ÷ control group PBMCs proliferation rate × 100%.

[0031] Comparative Example: Three P2 umbilical cord mesenchymal stem cells from the same donor as in the example were resuscitated from the cell bank by the following method: turning on a water bath and adjusting the temperature to 37° C.; placing the cells in a water bath for rapid rewarming for 3 minutes; and when all the cells were dissolved, resuscitation was complete.

[0032] After the cells were recovered, they were taken out and mixed with stem cell culture medium (αMEM medium + 5% human platelet lysate) and inoculated in 75cm 2 In the culture bottle, the inoculation density is 5000-6000 cells / cm 2 , cultured for 3-5 days at 37°C, 5.0% CO2, and the confluence reached 85-95%. The cells were harvested and were P3 cells.

[0033] P3 cells were mixed with stem cell culture medium and seeded again in 75 cm 2 In the culture bottle, the inoculation density is 5000-6000 cells / cm 2 , cultured for 3-5 days at 37°C, 5.0% CO2, and the confluence reached 85-95%. The cells were harvested and were P4 cells.

[0034] P4 cells were mixed with stem cell culture medium and seeded again in 75 cm 2 In the culture bottle, the inoculation density is 5000-6000 cells / cm 2 , cultured for 3-5 days at 37°C, 5.0% CO2, and when the confluence reached 85-95%, the cells were harvested as P5 cells.

[0035] Centrifuge the cells at 300g for 10 minutes, remove the supernatant, rinse the bottom of the culture container with PBS and remove it, add trypsin to digest the cells for 3 minutes, and then add 2 times the volume of culture medium to terminate the digestion process. Take out the cell suspension, add trypsin to the culture bottle to digest and harvest P5 cells. Resuspend the cell pellet in a freezing solution containing 5% DMSO, cool it down with a program cooling device, and store it in a liquid nitrogen tank.

[0036] After 3 days, the frozen cells were taken out and revived again, and the expression levels of IDO, ANG-1, NGF, HGF, and SOD were detected, and the ability to regulate T cell immunity was also tested.

[0037] Implementation effect example:

[0038] Twenty rats about 8 weeks old were selected and divided into a normal control group, an aging model group, a comparative example treatment group, and an example treatment group, with 5 rats in each group.

[0039] The normal group was injected with saline every day for 4 consecutive months; the other groups were injected with D-galactose every day for 4 consecutive months. Afterwards, the control group was injected with umbilical cord mesenchymal stem cells of the control group, and the example treatment group was injected with umbilical cord mesenchymal stem cells with enhanced anti-aging properties of the example, once a week for 4 consecutive weeks. The normal control group and the aging model group were injected with saline.

[0040] After 4 weeks of treatment, the serum of rats in all groups was collected to detect the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA). The activity of SOD was detected by xanthine oxidase reaction method. The content of MDA was determined by thiobarbituric acid colorimetry according to the instructions of the kit.

[0041] The mouse thymus tissue homogenate was centrifuged at 3000 r / min for 10 min, and the supernatant was taken to detect the IL-2 and IL-10 contents by ELISA method according to the instructions of the kit.

[0042] The IDO expression levels of umbilical cord mesenchymal stem cells in the comparative examples and embodiments of the present invention are as follows: Figure 1 As shown, it can be seen that the control example has almost no IDO expression, while the example example IDO expression is significantly increased.

[0043] The expression levels of ANG-1, NGF, HGF, and SOD of umbilical cord mesenchymal stem cells in the comparative examples and embodiments of the present invention are as follows: Figures 2 to 5 As shown, the expression of the example is higher than that of the control example, indicating that the mesenchymal stem cells of the example have better tissue repair, neural cell and antioxidant abilities.

[0044] The inhibition of lymphocyte proliferation by umbilical cord mesenchymal stem cells in the comparative examples and embodiments of the present invention is as follows: Figure 6 As shown, the embodiment has better immune regulation ability than the comparative example.

[0045] The anti-aging effect of the umbilical cord mesenchymal stem cells of the present invention is as follows Figures 7 to 9 As shown, compared with the comparative example, the expression of SOD in the serum of the treated rats in the embodiment was significantly increased, and the expression of MDA was significantly decreased, indicating that the umbilical cord mesenchymal stem cells in the embodiment can more effectively remove free radicals in the aging body. Compared with the comparative example, the IL-10 content in the thymus of the treated rats in the embodiment was significantly increased, indicating that the umbilical cord mesenchymal stem cells in the embodiment can better regulate the low-inflammatory state in the body.

[0046] The design focus of the present invention is that the mesenchymal stem cells obtained by adopting the mesenchymal stem cell production method for anti-aging provided by the present invention have better anti-aging effects, and the immune regulation ability and specific factor secretion level of mesenchymal stem cells are improved by simply adding cytokines, thereby establishing a method for producing mesenchymal stem cells for use in anti-aging related diseases; the method is highly operational and has significant effects; the enhanced anti-aging effect includes improving the low inflammatory state in the body caused by aging or obesity, thereby reducing the risks of related diseases such as diabetes and increased blood lipids.

[0047] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for producing mesenchymal stem cells for anti-aging, characterized in that: The steps include: S1. Cell culture: Mesenchymal stem cells were cultured in vitro until the cell confluence reached 60%-95%; S2, cytokine stimulation: add cytokines to the culture medium of the cells and stimulate for 24h-48h; S3. Detection and screening: digest and collect mesenchymal stem cells, detect the expression levels of indoleamine 2,3-dioxygenase (IDO) and related cytokines in mesenchymal stem cells, and detect the immune regulation ability of T cells. Increase the IDO expression level by more than 1,000 times to select mesenchymal stem cells for anti-aging applications.

2. The method for producing mesenchymal stem cells for anti-aging according to claim 1, characterized in that: The in vitro culturing of mesenchymal stem cells in step S1 is specifically as follows: the mesenchymal stem cells are cultured at a density of 5000-6000 cells / cm 2 The cells were inoculated at a high density and cultured in αMEM medium supplemented with 5% human platelet lysate at 37°C in a 5.0% CO2 incubator.

3. The method for producing mesenchymal stem cells for anti-aging according to claim 1, characterized in that: The concentration of the cytokines in step S2 is 10-50 ng / mL, and the cytokines include one or a combination of IFN-γ, TNF-α, IL-17A or IL-1β.

4. The method for producing mesenchymal stem cells for anti-aging according to claim 1, characterized in that: The digestion and collection of mesenchymal stem cells in step S3 are specifically as follows: first remove the culture supernatant, rinse the bottom of the culture container with PBS and then remove it, add trypsin to digest the cells for 3 minutes, and then add 2 times the volume of culture medium to terminate the digestion process; take out the cell suspension, centrifuge at 300g for 10 minutes, remove the supernatant, and collect the mesenchymal stem cells.

5. The method for producing mesenchymal stem cells for anti-aging according to claim 1, characterized in that: In step S3, the expression levels of indoleamine 2,3-dioxygenase (IDO) and related cytokines in mesenchymal stem cells are detected by qPCR method, and the related cytokines include ANG-1, NGF, HGF and SOD.

6. The method for producing mesenchymal stem cells for anti-aging according to claim 5, characterized in that: The qPCR method includes: RNA extraction: using TRIzol reagent to extract total RNA from mesenchymal stem cells; cDNA synthesis: using a reverse transcription kit to reverse transcribe the extracted RNA into cDNA; qPCR reaction: designing specific primers for IDO, ANG-1, NGF, HGF and SOD; preparing a qPCR reaction system: adding cDNA samples and performing real-time fluorescence quantitative PCR reaction; data analysis: calculating the relative expression amount by the 2^(-ΔΔCt) method, and using GAPDH as an internal reference gene for standardization.

7. The method for producing mesenchymal stem cells for anti-aging according to claim 1, characterized in that: In step S3, the T cell immune regulation ability is detected by a co-culture method, which includes: labeling peripheral blood mononuclear cells (PBMCs); then stimulating CFSE-labeled PBMCs with 10 ng / ml phytohemagglutinin, and co-culturing with umbilical cord mesenchymal stem cells at a ratio of 5:1 at 37°C and 5% CO2 for 4-5 days.

8. The method for producing mesenchymal stem cells for anti-aging according to claim 7, characterized in that: The peripheral blood mononuclear cells (PBMCs) are labeled with carboxyfluorofluorescein ethyl succinimidyl ester (CFSE).

9. The method for producing mesenchymal stem cells for anti-aging according to claim 7, characterized in that: After the co-culture was completed for 4-5 days, all lymphocytes were collected and CFSE fluorescence was detected by using a flow analyzer.

10. The method for producing mesenchymal stem cells for anti-aging according to claim 1, characterized in that: In step S1, the mesenchymal stem cells are derived from umbilical cord tissue, umbilical cord blood tissue, placental tissue, adipose tissue, bone marrow tissue or dental pulp tissue.

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