A high expansion capacity human adipose-derived mesenchymal stromal cell subpopulation and characteristics thereof

Abstract

The present application relates to the technical field of biology, in particular to a human adipose-derived mesenchymal stromal cell subpopulation with high expansion capacity and characteristics thereof. The human adipose-derived mesenchymal stromal cell provided by the present application has high expansion capacity, and can obtain more than 10 million therapeutic doses (each therapeutic dose refers to 50 million cells) from a single original source tissue, which can meet the required cell amount for treating a certain indication, and does not need to bear the risk of donor difference and clinical uncertainty involved in cells from different donors, so that the human adipose-derived mesenchymal stromal cell obtained by the preparation method of the present application is safer for clinical use.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a highly proliferative human adipose-derived mesenchymal stromal cell subpopulation and its characteristics. Background Technology

[0002] Mesenchymal stromal cells (MSCs) are cells widely distributed in various human tissues. Previous research has shown that these cells can be isolated from tissues such as bone marrow, umbilical cord, and adipose tissue. Due to their low immunogenicity and immunomodulatory functions, MSCs are currently used as cell therapy drugs in clinical research for the treatment of various diseases. However, many factors limit the clinical application of MSCs. One major reason is their limited in vitro expansion capacity. The low expansion capacity of human MSCs restricts cell production, making it difficult to meet the treatment needs of most indications, especially those requiring multiple injections to the same patient. To meet the required cell volume, it is necessary to use human MSCs from multiple donors, which not only increases ethical issues but also challenges the quality consistency of human MSC products and introduces safety risks to clinical treatment. Summary of the Invention

[0003] This invention aims to partially address the problem of insufficient in vitro expansion capacity of human adipose-derived mesenchymal stem cells (ADMSCs) in the field. To this end, this invention proposes an ADMSC with high expansion capacity and a method for its preparation. The ADMSCs obtained by this invention exhibit outstanding expansion capacity; after isolation from a single original source tissue, more than 10 million therapeutic doses can be obtained in vitro, sufficient to meet the cell quantity required for a specific indication, thereby avoiding donor variability and corresponding clinical uncertainties and risks caused by cells from different donor sources. The ADMSCs prepared by the method of this invention have higher safety for clinical use.

[0004] Therefore, a first aspect of the present invention provides adipose-derived mesenchymal stromal cells with high amplification capacity. In some embodiments of the present invention, compared with mesenchymal stromal cells from other tissue sources, the genes in the adipose-derived mesenchymal stromal cells are... NBL1, MASP1 High expression, and gene BUB1 , CDCA8 , PBK , PCDH10 Low expression.

[0005] In some embodiments of the present invention, a combination of highly expressed or poorly expressed genes is used to achieve either high or low expression in adipose-derived mesenchymal stromal cells (ADMSCs). Specifically, high expression refers to a ratio (log2 expression ratio) greater than 1.5 in transcriptomic sequencing results of mesenchymal stromal cells from various tissue sources, where the expression level of the gene in ADMSCs is higher than that in mesenchymal stromal cells from other tissue sources. Conversely, low expression refers to a ratio (log2 expression ratio) less than -1.5 in transcriptomic sequencing results of mesenchymal stromal cells from various tissue sources, where the expression level of the gene in ADMSCs is lower than that in mesenchymal stromal cells from other tissue sources.

[0006] In some embodiments of the present invention, the adipose-derived mesenchymal stromal cells have the following gene expression characteristics: The adipose-derived mesenchymal matrix cells cultured under optimal culture conditions contain genes within the cells. NFKBIZ, PHGDH SLC1A5, MMP1, CLU, NDVFA4L2, CPXM2 High expression, and gene FADS2, DHCR24, HMGCS1, SPARC, POSTN, MYBL2, KIF4A, KIF20A Low expression.

[0007] The aforementioned high-expression or low-expression genes are combined and expressed at high or low levels in adipose-derived mesenchymal matrix cells under optimal culture conditions. Specifically, high expression refers to a log2 expression ratio (the ratio of the gene's expression level under optimal culture conditions to that under other culture conditions) greater than 1.5 in transcriptomic sequencing results of adipose-derived mesenchymal matrix cells cultured under multiple culture conditions, and this is confirmed by enzyme-linked immunosorbent assay (ELISA) or quantitative polymerase chain reaction (PCR) experiments, indicating that the gene's content in adipose-derived mesenchymal matrix cells cultured under optimal conditions is higher than that in adipose-derived mesenchymal matrix cells cultured under other culture conditions. Low expression refers to a log2 expression ratio (the ratio of the gene's expression level under optimal culture conditions to that under other culture conditions) less than -1.5 in transcriptomic sequencing results of adipose-derived mesenchymal matrix cells cultured under multiple culture conditions, and this is confirmed by enzyme-linked immunosorbent assay (ELISA) or quantitative polymerase chain reaction (PCR) experiments, indicating that the gene's content in adipose-derived mesenchymal matrix cells cultured under optimal conditions is lower than that in adipose-derived mesenchymal matrix cells cultured under other culture conditions.

[0008] In some embodiments of the present invention, when genes in the cell NBL1, MASP1, NFKBIZ, PHGDH, SLC1A5, MMP1, CLU, NDVFA4L2, CPXM2 As a combination of highly expressed genes, and BUB1 , CDCA8 , PBK , PCDH10, FADS2 DHCR24, HMGCS1, SPARC, POSTN, MYBL2, KIF4A, KIF20A When expressed in combination at low levels, the cells are adipose-derived mesenchymal matrix cells cultured under optimal conditions with high amplification capacity.

[0009] It is precisely due to the synergistic expression of these genes that the adipose-derived mesenchymal stromal cells provided by this invention possess unique functions, namely, strong proliferative capacity, enabling the acquisition of over 10 million therapeutic doses from a single original tissue source. When these genes are simultaneously highly expressed in cells... NBL1, MASP1, NFKBIZ, PHGDH, SLC1A5, MMP1, CLU, NDVFA4L2, CPXM2 and low expression BUB1 , CDCA8 , PBK , PCDH10, FADS2, DHCR24, HMGCS1, SPARC, POSTN, MYBL2, KIF4A, KIF20A At that time, the cell was the adipose-derived mesenchymal matrix cell with high amplification capacity cultured under optimal conditions.

[0010] In some embodiments of the present invention, the adipose-derived mesenchymal stromal cells are obtained by culturing them using the following methods: S1: Adipose-derived mesenchymal matrix cells were isolated from the donor fat; S2: The adipose-derived mesenchymal stromal cells were cultured using a complete culture medium. The complete culture medium includes a basal culture medium and additives, the additives including serum, non-essential amino acids, epidermal growth factor, insulin-like growth factor 1, and basic fibroblast growth factor. In step S2, the cell culture is carried out under 3-7% O2 conditions. Preferably, the cell culture in step S2 is carried out under 3% O2 ​​conditions.

[0011] The highly proliferative adipose-derived mesenchymal stromal cells provided by this invention reduce the batch-to-batch variability and instability of human adipose-derived mesenchymal stromal cells as a drug. More than 10 million therapeutic doses can be obtained using a single donor source, which can meet the cell quantity required for the treatment of a certain indication.

[0012] In some embodiments of the invention, the working concentration of serum in the complete culture medium is 10%-25%. Preferably, the working concentration of serum is 20%.

[0013] In some embodiments of the invention, the working concentration of the non-essential amino acids in the complete culture medium is 1%.

[0014] In some embodiments of the present invention, the working concentration of the epidermal growth factor in the complete culture medium is 2-10 ng / mL.

[0015] In some embodiments of the present invention, the working concentration of insulin-like growth factor 1 in the complete culture medium is 2-10 ng / mL.

[0016] In some embodiments of the present invention, the working concentration of the basic fibroblast growth factor is 5-20 ng / mL. Preferably, it is 16 ng / mL.

[0017] In some embodiments of the present invention, the basal culture medium is selected from at least one of DMEM α, DMEM / F12, DMEM high glucose, Ham's F-12, and RIM-1640.

[0018] In some embodiments of the present invention, the basal culture medium is DMEM α.

[0019] In some embodiments of the present invention, the seeding density of the adipose-derived mesenchymal matrix cells during cell culture is 350-800 cells / mL. In some embodiments of the present invention, the cell culture temperature is 35-38 ℃ and the culture time is 5-8 days.

[0020] In some embodiments of the invention, the donor is a mammal, such as a human, monkey, dog, or rabbit, but preferably a human.

[0021] A second aspect of the present invention provides a method for preparing the highly proliferative adipose-derived mesenchymal stromal cells described in the first aspect. In some embodiments of the present invention, the method includes: S1: Adipose-derived mesenchymal matrix cells were isolated from the donor fat; S2: The adipose-derived mesenchymal stromal cells were cultured using a complete culture medium. The complete culture medium includes a basal culture medium and additives, the additives including serum, non-essential amino acids, epidermal growth factor, insulin-like growth factor 1, and basic fibroblast growth factor. In step S2, the cells are cultured under 3-7% O2 conditions.

[0022] The method for preparing highly expandable adipose-derived mesenchymal stem cells (ADMSCs) provided by this invention improves the in vitro expansion capacity and yield of ADMSCs, and ensures stable passage for at least 10 generations. Existing methods for producing ADMSCs have low expansion capacity; for example, human ADMSCs isolated from the same donor cannot meet the cell quantity required for a specific indication after expansion, necessitating the isolation of human ADMSCs from multiple donors. This increases the instability and variability of human ADMSCs as a drug. This invention solves this problem, increasing the therapeutic dose obtained from a single original tissue source from only a few hundred to a few thousand units to over 10 million units, sufficient to meet the cell quantity required for a specific indication, and making clinical use safer.

[0023] In some embodiments of the invention, the working concentration of serum in the complete culture medium is 10%-25%. Preferably, the working concentration of serum is 20%.

[0024] In some embodiments of the invention, the working concentration of the non-essential amino acids in the complete culture medium is 1%.

[0025] In some embodiments of the present invention, the working concentration of the epidermal growth factor in the complete culture medium is 2-10 ng / mL.

[0026] In some embodiments of the present invention, the working concentration of insulin-like growth factor 1 in the complete culture medium is 2-10 ng / mL.

[0027] In some embodiments of the present invention, the working concentration of the basic fibroblast growth factor is 5-20 ng / mL. Preferably, it is 16 ng / mL.

[0028] In some embodiments of the present invention, the basal culture medium is selected from at least one of DMEM α, DMEM / F12, DMEM high glucose, Ham's F-12, and RIM-1640.

[0029] In some embodiments of the present invention, the basal culture medium is DMEM α.

[0030] In some embodiments of the present invention, the seeding density of the adipose-derived mesenchymal matrix cells during cell culture is 350-800 cells / mL. In some embodiments of the present invention, the cell culture temperature is 35-38 ℃ and the culture time is 5-8 days.

[0031] In some embodiments of the invention, the donor is a mammal, such as a human, monkey, dog, or rabbit, but preferably a human.

[0032] A third aspect of the present invention provides a pharmaceutical composition. In some embodiments of the present invention, the pharmaceutical composition comprises the highly amplifying adipose-derived mesenchymal matrix cells described in the first aspect or the highly amplifying adipose-derived mesenchymal matrix cells prepared by the method described in the second aspect.

[0033] A fourth aspect of the present invention provides passaged cells. In some embodiments of the present invention, the cells are obtained by passage of highly proliferative adipose-derived mesenchymal stem cells as described in the first aspect; Alternatively, it can be obtained by passage of highly amplifying adipose-derived mesenchymal matrix cells prepared by the method described in the second aspect.

[0034] In some embodiments of the present invention, the PDL (Population Doubling Level) does not exceed 40.

[0035] The fifth aspect of this invention provides the use of the highly proliferative adipose-derived mesenchymal stem cells described in the first aspect, the highly proliferative adipose-derived mesenchymal stem cells prepared by the method described in the second aspect, and the passaged cells described in the fourth aspect in the preparation of pharmaceutical compositions. In some embodiments of this invention, the pharmaceutical compositions are used for cell therapy.

[0036] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0037] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The study demonstrated the use of quantitative real-time polymerase chain reaction (qPCR) to detect the presence of certain substances in human adipose mesenchymal stromal cells (hADMSCs) under culture conditions 1 and 2. FADS2 , DHCR24 , HMGCS1 The expression is as follows. Among them, **** represents... p <0.0001; Figure 2 The results show the detection of hADMSCs under culture conditions 1 and 2 using qPCR. NFKBIZ , PHGDH , SLC1A5 The expression is as follows, where **** represents p<0.0001; Figure 3 This demonstrates the detection of hADMSCs under culture conditions 1 and 3 using enzyme-linked immunosorbent assays (ELISA). SPARC and POSTN The expression situation; Figure 4 The results show the detection of hADMSCs under culture conditions 1 and 3 using ELISA. MMP1 and CLU The expression situation; Figure 5 The results show the detection of hADMSCs under culture conditions 1 and 4 using qPCR. MYBL2 , KIF4A , KIF20A The expression is as follows, where **** represents p <0.0001; Figure 6 The results show the detection of hADMSCs under culture conditions 1 and 4 using qPCR. NDVFA4L2 , CPXM2 The expression is as follows, where ** represents p <0.01, **** represents p <0.0001; Figure 7 The gene was shown to be highly expressed in hADMSCs compared to the other three types of mesenchymal stromal cells. NBL1 The qPCR test results, where **** represents p <0.0001, hBMMSC is human bone marrow mesenchymal stromal cell, hUCMSC is human umbilical cord mesenchymal stromal cell, and hPLMSC is human placental mesenchymal stromal cell; Figure 8 The gene was shown to be highly expressed in hADMSCs compared to the other three types of mesenchymal stromal cells. MASP1 The ELISA results showed that hBMMSCs were human bone marrow mesenchymal stromal cells, hUCMSCs were human umbilical cord mesenchymal stromal cells, and hPLMSCs were human placental mesenchymal stromal cells. Figure 9 The gene was shown to be expressed at low levels in hADMSCs compared to the other three types of mesenchymal stromal cells. BUB1 , CDCA8 , PBK , PCDH10 The qPCR test results, where * represents p <0.05, ** represents p <0.01, *** represents p <0.001, **** representsp <0.0001, hBMMSC is human bone marrow mesenchymal stromal cell, hUCMSC is human umbilical cord mesenchymal stromal cell, and hPLMSC is human placental mesenchymal stromal cell; Figure 10 The growth curves of hADMSCs after continuous passage under different culture conditions are shown. Figure 11 This shows the theoretical therapeutic amount of hADMSCs that can be expanded under optimal culture conditions (condition 1); Figure 12 The results of hADMSC formation under culture conditions 1, 2, 3, and 4 are shown. * indicates... p <0.05, ** represents p <0.01, **** represents p <0.0001. Detailed Implementation

[0038] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0039] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0040] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0041] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0043] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0044] According to a specific embodiment of the present invention, the present invention provides adipose-derived mesenchymal stromal cells with high amplification capacity. Compared with mesenchymal stromal cells from other tissue sources, the genes in the adipose-derived mesenchymal stromal cells are... NBL1, MASP1 High expression, and gene BUB1 , CDCA8 , PBK , PCDH10 Low expression.

[0045] The mesenchymal stromal cells from other tissue sources include, but are not limited to, human mesenchymal stromal cells derived from human bone marrow, human umbilical cord, and human placenta.

[0046] According to a specific embodiment of the present invention, the adipose-derived mesenchymal stromal cells have the following gene expression characteristics: The adipose-derived mesenchymal matrix cells cultured under optimal culture conditions contain genes within the cells. NFKBIZ, PHGDH SLC1A5, MMP1, CLU, NDVFA4L2, CPXM2 High expression, and gene FADS2, DHCR24, HMGCS1, SPARC, POSTN, MYBL2, KIF4A, KIF20A Low expression.

[0047] The “optimal culture conditions” refer to the conditions most suitable for the growth and proliferation of the adipose-derived mesenchymal matrix cells. In this document, it refers to the following culture conditions: cultured in a medium containing basal medium, serum, non-essential amino acids, epidermal growth factor, insulin-like growth factor 1, and basic fibroblast growth factor, with culture conditions of 3-7% O2, a culture temperature of 35-38 ℃, and a culture time of 5-8 days.

[0048] According to a specific embodiment of the present invention, the basal culture medium in the culture medium with optimal culture conditions includes, but is not limited to, at least one of DMEM α, DMEM / F12, DMEM high glucose, Ham's F-12, and RIM-1640.

[0049] According to a specific embodiment of the present invention, the working concentration of serum in the culture medium under optimal culture conditions is 10%-25%; the working concentration of the non-essential amino acids is 1%; the working concentration of epidermal growth factor is 2-10 ng / mL; the working concentration of insulin-like growth factor 1 is 2-10 ng / mL; and the working concentration of basic fibroblast growth factor is 5-20 ng / mL.

[0050] According to a specific embodiment of the present invention, when genes in the cell NBL1, MASP1, NFKBIZ, PHGDH, SLC1A5, MMP1, CLU, NDVFA4L2, CPXM2 As a combination of highly expressed genes, and BUB1 , CDCA8 , PBK , PCDH10 FADS2, DHCR24, HMGCS1, SPARC, POSTN, MYBL2, KIF4A, KIF20A When expressed in combination at low levels, the cells are adipose-derived mesenchymal matrix cells cultured under optimal conditions with high amplification capacity.

[0051] According to a specific embodiment of the present invention, the adipose-derived mesenchymal stromal cells are obtained by culturing using the following method: S1: Adipose-derived mesenchymal matrix cells were isolated from the donor fat; S2: The adipose-derived mesenchymal stromal cells were cultured using a complete culture medium. The complete culture medium includes a basal culture medium and additives, the additives including serum, non-essential amino acids, epidermal growth factor, insulin-like growth factor 1, and basic fibroblast growth factor. In step S2, the cells are cultured under 3-7% O2 conditions.

[0052] According to a specific embodiment of the present invention, the working concentration of serum in the complete culture medium is 10%-25%, for example, the working concentration of serum can be 10%, 15%, 20%, 25%, etc. According to a specific embodiment of the present invention, the working concentration of the non-essential amino acids in the complete culture medium is 1%.

[0053] According to a specific embodiment of the present invention, the working concentration of the epidermal growth factor in the complete culture medium is 2-10 ng / mL. For example, the working concentration of the epidermal growth factor can be 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, or 10 ng / mL.

[0054] According to a specific embodiment of the present invention, the working concentration of insulin-like growth factor 1 in the complete culture medium is 2-10 ng / mL; for example, the working concentration of insulin-like growth factor 1 is 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, or 10 ng / mL.

[0055] According to a specific embodiment of the present invention, the working concentration of the basic fibroblast growth factor in the complete culture medium is 5-20 ng / mL, for example, the working concentration of the basic fibroblast growth factor is 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, etc.

[0056] According to one specific embodiment of the present invention, the basal culture medium is selected from at least one of DMEM α, DMEM / F12, DMEM high glucose, Ham's F-12, and RIM-1640. According to a preferred embodiment of the present invention, the basal culture medium is DMEM α.

[0057] According to a specific embodiment of the present invention, the seeding density of the adipose-derived mesenchymal matrix cells during cell culture is 350-800 cells / mL. For example, the seeding density of the adipose-derived mesenchymal matrix cells can be 350 cells / mL, 400 cells / mL, 450 cells / mL, 500 cells / mL, 550 cells / mL, 600 cells / mL, 650 cells / mL, 700 cells / mL, 750 cells / mL, 800 cells / mL, etc.

[0058] According to one specific embodiment of the present invention, the cell culture temperature is 35-38 °C and the culture time is 5-8 days. According to a preferred embodiment of the present invention, the cell culture temperature is 37 °C and the culture time is 7 days.

[0059] According to a specific embodiment of the present invention, the present invention provides a method for preparing the aforementioned highly proliferative adipose-derived mesenchymal stromal cells, comprising: S1: Adipose-derived mesenchymal matrix cells were isolated from the donor fat; S2: The adipose-derived mesenchymal stromal cells were cultured using a complete culture medium. The composition of the complete culture medium and the conditions for cell culture are as described above.

[0060] According to one specific embodiment of the present invention, the donor is a mammal.

[0061] According to a specific embodiment of the present invention, the present invention also provides a pharmaceutical composition comprising the aforementioned highly expandable adipose-derived mesenchymal stromal cells or the highly expandable adipose-derived mesenchymal stromal cells prepared by the aforementioned method. It should be noted that the pharmaceutical composition of the present invention may contain other pharmaceutical excipients in addition to adipose-derived mesenchymal stromal cells, and these formulations are all covered within the scope of protection of the present invention.

[0062] According to a specific embodiment of the present invention, the present invention also provides a passaged cell, which is obtained by passage of the aforementioned highly proliferative adipose-derived mesenchymal matrix cells. Alternatively, it can be obtained by passage of highly amplifying adipose-derived mesenchymal stromal cells prepared by the methods described above.

[0063] According to a specific embodiment of the present invention, the Population Doubling Level (PDL) does not exceed 40.

[0064] According to a specific embodiment of the present invention, the present invention also provides the use of the aforementioned highly proliferative adipose-derived mesenchymal stromal cells, the highly proliferative adipose-derived mesenchymal stromal cells prepared by the aforementioned method, and the aforementioned passaged cells in the preparation of a pharmaceutical composition, said pharmaceutical composition being used for cell therapy.

[0065] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0066] Example 1: Obtaining P0 generation human adipose-derived mesenchymal stromal cells First, the adipose tissue was washed by adding sufficient DPBS and thoroughly agitating and washing. After standing and separating into layers, the lower layer was discarded. This process was repeated 3–5 times until the supernatant was clear and colorless. Then, collagenase digestion was performed by adding type I collagenase working solution at a 1:1 ratio to the fat volume. The mixture was incubated at 37 ℃ for 45–60 min, gently mixing every 10 min until the adipose tissue became emulsified. After digestion, an equal volume of MSC complete culture medium (DMEM α + 20% fetal bovine serum + 1% non-essential amino acids + 5 ng / mL epidermal growth factor + 5 ng / mL insulin-like growth factor 1 + 16 ng / mL basic fibroblast growth factor) was added to terminate the digestion. The mixture was gently agitated and mixed. The cell suspension was filtered through a 100 μm cell sieve to remove undigested tissue. The filtrate was centrifuged at 1200×g for 10 minutes. Centrifuge for 5 min, discarding the upper lipid and supernatant, retaining the basal stromal vascular layer (SVF) cell pellet; then resuspend the pellet in complete culture medium, centrifuge at 500×g for 5 min, discard the supernatant, and resuspend the pellet again in complete culture medium to complete cell counting and viability testing; subsequently, perform primary seeding at 20,000 cells / cm³. 2Cells were seeded into T75 culture flasks, mixed, and incubated at 37 ℃ in a 3% O2 ​​incubator. The medium was changed for the first time after 24 h, and then every 48 h thereafter. When the cell confluence reached 70%–80%, the cells were harvested. The original medium was discarded, and the cell surface was washed 1–2 times with HBSS. 3 mL of TrypLE™ Express digestion solution was added, and the cells were digested at 37 ℃ for 1–3 min. After observing under a microscope until the cells became round and the intercellular spaces increased, 6 mL of complete culture medium was added to stop the digestion. The cell suspension was collected by gently blowing on the flask wall with a pipette, transferred to a centrifuge tube, and centrifuged at 500×g for 5 min. The supernatant was discarded, and the cell pellet was resuspended in complete culture medium. Cell counting was performed again. The cells collected at this time were P0 generation human adipose mesenchymal matrix cells.

[0067] Example 2: Comparison of gene expression in human adipose-derived mesenchymal stromal cells under different culture conditions The following groups of human adipose mesenchymal stromal cells (hADMSCs) were obtained under different culture conditions, and cell growth curves were plotted under each culture condition. Cell transcriptome sequencing was performed on cells from the same passage under different culture conditions, and the sequencing results of the selected genes were verified. Monoclonal formation experiments were conducted on cells from the same passage under different culture conditions.

[0068] 1. Design of different culture conditions Equal amounts of P0 generation cells were seeded into culture dishes and subjected to continuous in vitro passage culture according to the following grouping conditions. The P0 generation cells were obtained using the same method as described in Example 1.

[0069] Equal amounts of P0 generation cells were seeded into culture dishes and continuously passaged in vitro according to the following grouping conditions.

[0070] (1) The culture medium used in Condition 1 consisted of DMEM α + 20% fetal bovine serum + 1% non-essential amino acids + 5 ng / mL epidermal growth factor + 5 ng / mL insulin-like growth factor 1 + 16 ng / mL basic fibroblast growth factor. The culture conditions were 37 ℃, 3% O2, and cultured for 7 days. The culture medium used under these conditions was recorded as complete culture medium, and the culture conditions were recorded as optimal culture conditions.

[0071] (2) The culture medium used in condition 2 consisted of DMEM α + 5% fetal bovine serum + 1% non-essential amino acids + 5 ng / mL epidermal growth factor + 5 ng / mL insulin-like growth factor 1 + 16 ng / mL basic fibroblast growth factor. The culture conditions were 37 ℃, 3% O2, and cultured for 7 days. (3) The culture medium used in condition 3 consisted of DMEM α + 20% fetal bovine serum + 1% non-essential amino acids + 5 ng / mL epidermal growth factor + 5 ng / mL insulin-like growth factor 1, and the culture conditions were 37 ℃, 3% O2, and cultured for 7 days. (4) The culture medium used in condition 4 consisted of DMEM α + 20% fetal bovine serum + 1% non-essential amino acids + 5 ng / mL epidermal growth factor + 5 ng / mL insulin-like growth factor 1 + 16 ng / mL basic fibroblast growth factor. The culture conditions were 37 ℃, 19% O2, and cultured for 7 days.

[0072] 2. Gene expression in human adipose-derived mesenchymal matrix cells under different culture conditions (1) Expression of differentially expressed genes in hADMSCs under culture conditions 1 and 2 ① Detection of genes with low expression in hADMSCs cultured under condition 1 compared to culture condition 2 Figure 1 For qPCR detection of hADMSCs under culture conditions 1 and 2 FADS2 , DHCR24 , HMGCS1 The expression status of [the substance / factor]. qPCR validation data confirmed that, compared to culture condition 2, hADMSCs cultured under condition 1 showed [a decrease in expression / value]. FADS2 , DHCR24 , HMGCS1 The level of expression decreased.

[0073] ② Compared with culture condition 2, the genes highly expressed in hADMSCs cultured under condition 1 Figure 2 For qPCR detection of hADMSCs under culture conditions 1 and 2 NFKBIZ , PHGDH , SLC1A5 The expression status of [the substance / factor]. qPCR validation data confirmed that, compared to culture condition 2, hADMSCs cultured under condition 1 showed [a decrease in expression / value]. NFKBIZ , PHGDH , SLC1A5 The expression level increased.

[0074] In summary, and combined with the experimental results from other embodiments, it can be seen that hADMSCs cultured under condition 1 are a group of human adipose-derived mesenchymal stromal cells with high proliferative capacity. Combined with gene expression verification results, the gene expression characteristics of this group of hADMSCs are as follows: FADS2 low , DHCR24 low , HMGCS1 low NFKBIZ high , PHGDH high , SLC1A5 high .

[0075] (2) Expression of differentially expressed genes in hADMSCs under culture conditions 1 and 3 ① Detection of genes with low expression in hADMSCs cultured under condition 1 compared to culture condition 3. Figure 3 For ELISA detection of hADMSCs under culture conditions 1 and 3 SPARC and POSTN The expression status of hADMSCs. ELISA validation data confirmed that, compared to condition 3, under condition 1, hADMSCs showed... SPARC and POSTN The level of expression decreased.

[0076] ② Detection of highly expressed genes in hADMSCs cultured under condition 1 compared to culture condition 3. Figure 4 For ELISA detection of hADMSCs under culture conditions 1 and 3 MMP1 and CLU The expression status of hADMSCs was confirmed by ELISA validation data, showing that hADMSCs cultured under condition 1 had significantly lower expression levels compared to culture condition 3. MMP1 and CLU The expression level increased.

[0077] In summary, and combined with the experimental results from other embodiments, it can be seen that hADMSCs cultured under condition 1 are a group of human adipose-derived mesenchymal stromal cells with high proliferative capacity. Combined with gene expression verification results, the gene expression characteristics of this group of hADMSCs are as follows: SPARC low , POSTN low MMP1 high , CLU high .

[0078] (3) Expression of differentially expressed genes in hADMSCs under culture conditions 1 and 4 ① Detection of genes with low expression in hADMSCs cultured under condition 1 compared to culture condition 4. Figure 5 For qPCR detection of hADMSCs under culture conditions 1 and 4 MYBL2 , KIF4A , KIF20A The expression status of hADMSCs was confirmed by qPCR validation data, which showed that, compared with culture condition 4, hADMSCs under condition 1 showed significantly lower expression levels. MYBL2 , KIF4A , KIF20A The level of expression decreased.

[0079] ② Detection of highly expressed genes in hADMSCs cultured under condition 1 compared to culture condition 4. Figure 6 For qPCR detection of hADMSCs under culture conditions 1 and 4 NDVFA4L2 , CPXM2 The expression status of [specific cells / elements]. qPCR validation data confirmed that, compared to culture condition 4, hADMSCs cultured under condition 1 showed [significantly lower expression levels]. NDVFA4L2 , CPXM2 The expression level increased.

[0080] In summary, and combined with the experimental results from other embodiments, it can be seen that hADMSCs under condition 1 are a group of human adipose-derived mesenchymal stromal cells with high proliferative capacity. Combined with gene expression verification results, the gene expression characteristics of this group of hADMSCs are as follows: MYBL2 low , KIF4A low , KIF20A low , NDVFA4L2 high , CPXM2 high .

[0081] (4) Summary Under optimal culture conditions (condition 1), adipose-derived MSCs exhibit stronger proliferative capacity than cells cultured under other conditions. In terms of gene expression, adipose-derived MSCs cultured under optimal conditions show high gene expression. NFKBIZ, PHGDH, SLC1A5 MMP1, CLU, NDVFA4L2, CPXM2 Gene combination, low expression FADS2, DHCR24, HMGCS1, SPARC, POSTN, MYBL2, KIF4A, KIF20A Gene combination.

[0082] Example 3: Detection of gene expression in human adipose-derived mesenchymal stromal cells. qPCR test results as follows Figure 7 and Figure 9 As shown, the ELISA test results are as follows: Figure 8As shown, hADMSCs exhibit higher gene expression compared to mesenchymal stromal cells from other tissue sources (including human bone marrow mesenchymal stromal cells hBMMSCs, human umbilical cord mesenchymal stromal cells hUCMSCs, and human placental mesenchymal stromal cells hPLMSCs). NBL1, MASP1 Gene, low expression BUB1 , CDCA8 , PBK , PCDH10 Gene.

[0083] Example 4: In vitro expansion capacity of human adipose-derived mesenchymal stromal cells (1) Growth curves of hADMSCs under different culture conditions Growth curves of hADMSCs after continuous passage under different culture conditions are as follows: Figure 10 As shown, hADMSCs cultured under condition 2 grow slower than those cultured under condition 1, hADMSCs cultured under condition 3 grow next, and hADMSCs cultured under condition 4 grow the slowest.

[0084] (2) The theoretical amplification of hADMSCs under optimal conditions Based on the existing stockpile of P0 hADMSCs and the cell growth kinetic parameters obtained from each passage and harvest, the final product cell quantity of all P0 hADMSCs cultured under optimal conditions was calculated. The results are as follows: Figure 11 As shown in Table 1, the amplification capacity of hADMSCs under the inventor's culture process and that of other companies is compared. The inventor's optimal culture process exhibits superior hADMSC amplification capacity compared to other companies, with a therapeutic dose of 11.25 million units (each therapeutic dose refers to 50 million cells).

[0085] Table 1

[0086] (3) Monoclonal formation ability of hADMSCs under different culture conditions The results of hADMSC monoclonal formation ability under different culture conditions are as follows: Figure 12 As shown in the results, compared with other culture conditions, hADMSCs cultured under the optimal culture condition showed the strongest single-clone formation ability, while hADMSCs cultured under condition 3 showed the weakest single-clone formation ability.

[0087] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0088] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A type of adipose-derived mesenchymal stromal cell with high proliferative capacity, characterized in that, Compared to mesenchymal stromal cells from other tissues, the adipose-derived mesenchymal stromal cells have higher gene density. NBL1, MASP1 High expression, and gene BUB1 , CDCA8 , PBK , PCDH10 Low expression.

2. The adipose-derived mesenchymal stromal cells according to claim 1, characterized in that, The adipose-derived mesenchymal stromal cells have the following gene expression characteristics: The adipose-derived mesenchymal matrix cells cultured under optimal culture conditions contain genes within the cells. NFKBIZ, PHGDH SLC1A5, MMP1, CLU, NDVFA4L2, CPXM2 High expression, and gene FADS2, DHCR24, HMGCS1, SPARC, POSTN, MYBL2, KIF4A, KIF20A Low expression.

3. The adipose-derived mesenchymal stromal cells according to claim 1, characterized in that, When genes in cells NBL1, MASP1, NFKBIZ, PHGDH, SLC1A5, MMP1, CLU, NDVFA4L2, CPXM2 As a combination of highly expressed genes, and BUB1 , CDCA8 , PBK , PCDH10, FADS2, DHCR24, HMGCS1, SPARC, POSTN, MYBL2, KIF4A, KIF20A When expressed in combination at low levels, the cells are adipose-derived mesenchymal matrix cells cultured under optimal conditions with high amplification capacity.

4. The adipose-derived mesenchymal stromal cells according to claim 1, characterized in that, The adipose-derived mesenchymal stromal cells were obtained by culturing them using the following method: S1: Adipose-derived mesenchymal matrix cells were isolated from the donor fat; S2: The adipose-derived mesenchymal stromal cells were cultured using a complete culture medium. The complete culture medium includes a basal culture medium and additives, the additives including serum, non-essential amino acids, epidermal growth factor, insulin-like growth factor 1, and basic fibroblast growth factor. In step S2, the cells are cultured under 3-7% O2 conditions.

5. The adipose-derived mesenchymal stromal cells according to claim 4, characterized in that, In the complete culture medium, the working concentration of serum is 10%-25%; Optionally, the working concentration of the non-essential amino acid is 1%; Optionally, the working concentration of the epidermal growth factor is 2-10 ng / mL; Optionally, the working concentration of insulin-like growth factor 1 is 2-10 ng / mL; Optionally, the working concentration of the basic fibroblast growth factor is 5-20 ng / mL.

6. The adipose-derived mesenchymal stromal cells according to claim 4, characterized in that, The basal culture medium is selected from at least one of DMEMα, DMEM / F12, DMEM high glucose, Ham's F-12, and RIM-1640; Optionally, the basal culture medium is DMEM α.

7. The adipose-derived mesenchymal stromal cells according to claim 4, characterized in that, When performing cell culture, the seeding density of the adipose-derived mesenchymal matrix cells is 350-800 cells / mL.

8. The adipose-derived mesenchymal stromal cells according to claim 4, characterized in that, When culturing cells, the culture temperature is 35-38 ℃ and the culture time is 5-8 days.

9. The adipose-derived mesenchymal matrix cells according to claim 4, characterized in that, The donor is a mammal.

10. A method for preparing adipose-derived mesenchymal stromal cells with high amplification capacity according to any one of claims 1-9, characterized in that, include: S1: Adipose-derived mesenchymal matrix cells were isolated from the donor fat; S2: The adipose-derived mesenchymal stromal cells were cultured using a complete culture medium. The complete culture medium includes a basal culture medium and additives, the additives including serum, non-essential amino acids, epidermal growth factor, insulin-like growth factor 1, and basic fibroblast growth factor. In step S2, the cells are cultured under 3-7% O2 conditions.

11. The method according to claim 10, characterized in that, In the complete culture medium, the working concentration of serum is 10%-25%; Optionally, the working concentration of the non-essential amino acid is 1%; Optionally, the working concentration of the epidermal growth factor is 2-10 ng / mL; Optionally, the working concentration of insulin-like growth factor 1 is 2-10 ng / mL; Optionally, the working concentration of the basic fibroblast growth factor is 5-20 ng / mL.

12. The method according to claim 10, characterized in that, The basal culture medium is selected from at least one of DMEM α, DMEM / F12, DMEM high glucose, Ham's F-12, and RIM-1640; Optionally, the basal culture medium is DMEM α; Optionally, when performing cell culture, the seeding density of the adipose-derived mesenchymal matrix cells is 350-800 cells / mL.

13. The method according to claim 10, characterized in that, When culturing cells, the culture temperature is 35-38 ℃ and the culture time is 5-8 days; Optionally, the donor is a mammal.

14. A pharmaceutical composition, characterized in that, It includes adipose-derived mesenchymal matrix cells with high amplification capacity as described in any one of claims 1-9 or adipose-derived mesenchymal matrix cells with high amplification capacity prepared by the method described in any one of claims 10-13.

15. A passaged cell, characterized in that, Obtained by passage of adipose-derived mesenchymal stromal cells with high amplification capacity as described in any one of claims 1-9; Alternatively, it can be obtained by passage of highly amplifying adipose-derived mesenchymal stromal cells prepared by any one of claims 10-13.

16. The cell according to claim 15, characterized in that, PDL should not exceed 40.

17. The use of the highly amplifying adipose-derived mesenchymal stem cells according to any one of claims 1-9, the highly amplifying adipose-derived mesenchymal stem cells prepared by the method according to any one of claims 10-13, and the passaged cells according to claim 15 or 16 in the preparation of a pharmaceutical composition, characterized in that, The pharmaceutical composition is used for cell therapy.

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