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CN111139221B - Culture and cryopreservation method of amniotic mesenchymal stem cells - Google Patents

  • ️Tue May 10 2022

CN111139221B - Culture and cryopreservation method of amniotic mesenchymal stem cells - Google Patents

Culture and cryopreservation method of amniotic mesenchymal stem cells Download PDF

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Publication number
CN111139221B
CN111139221B CN202010022849.4A CN202010022849A CN111139221B CN 111139221 B CN111139221 B CN 111139221B CN 202010022849 A CN202010022849 A CN 202010022849A CN 111139221 B CN111139221 B CN 111139221B Authority
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China
Prior art keywords
mesenchymal stem
stem cells
amniotic
culture
cells
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2020-01-09
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CN202010022849.4A
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CN111139221A (en
Inventor
李哲浩
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Sereno Beijing Biotechnology Co ltd
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Sereno Beijing Biotechnology Co ltd
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2020-01-09
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2020-01-09
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2022-05-10
2020-01-09 Application filed by Sereno Beijing Biotechnology Co ltd filed Critical Sereno Beijing Biotechnology Co ltd
2020-01-09 Priority to CN202010022849.4A priority Critical patent/CN111139221B/en
2020-05-12 Publication of CN111139221A publication Critical patent/CN111139221A/en
2022-05-10 Application granted granted Critical
2022-05-10 Publication of CN111139221B publication Critical patent/CN111139221B/en
Status Active legal-status Critical Current
2040-01-09 Anticipated expiration legal-status Critical

Links

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract

The invention relates to a culture and cryopreservation method of amniotic mesenchymal stem cells, which comprises amniotic tissue separation, amniotic mesenchymal stem cell separation, P0 generation amniotic mesenchymal stem cell culture and amplification culture. According to the culture method of the amniotic mesenchymal stem cells, in the separation stage of the amniotic mesenchymal stem cells, the amniotic mesenchymal stem cells can be more effectively separated from the amniotic tissues by digesting with a special mixed enzyme digestive juice system (the final concentration of each component in the digestive juice is 1.5-2U/mL neutral protease, 0.5mg/mL deoxyribonuclease I and 1mg/mL collagenase IV), so that the yield of the P0 generation cells is obviously improved.

Description

一种羊膜间充质干细胞的培养及冻存方法A kind of culture and cryopreservation method of amniotic mesenchymal stem cells

技术领域technical field

本发明属于干细胞培养技术领域，具体涉及一种羊膜间充质干细胞的培养及冻存方法。The invention belongs to the technical field of stem cell culture, and in particular relates to a culture and cryopreservation method of amniotic membrane mesenchymal stem cells.

背景技术Background technique

近年来随着对间充质干细胞功能特性的深入研究，间充质干细胞已经成为细胞替代治疗的研究热点。而人羊膜间充质干细胞除与应用广的骨髓间充质干细胞样具有多向分化潜能、免疫调节、造血支持等特性外，还具有来源广泛，取材方便，无伦理限制，细胞增殖能力强等优势。在各种组织器官中存活率高，存活时间长。源自胎盘羊膜组织的羊膜间充质干细胞是具有明显可塑性和多向分化潜能的一种成体干细胞，在不同生长因子的调节下，可分化成来源于2个胚层的不同组织细胞类型，如脂肪细胞、成骨细胞、肝样细胞、心肌样细胞、神经胶质细胞等，HAMSC不表达主要组织相容性II类抗原，微量表达主要组织相容性I类抗原，免疫原性低，移植排斥作用小，而且具有免疫调节功效，在干细胞治疗、组织工程治疗和再生医学研究应用中具有重要意义。In recent years, with the in-depth study of the functional properties of mesenchymal stem cells, mesenchymal stem cells have become a research hotspot in cell replacement therapy. Human amniotic mesenchymal stem cells, in addition to the widely used bone marrow mesenchymal stem cells, have the characteristics of multi-directional differentiation potential, immune regulation, and hematopoietic support. Advantage. It has high survival rate and long survival time in various tissues and organs. Amniotic mesenchymal stem cells derived from placental amniotic tissue are adult stem cells with obvious plasticity and multi-directional differentiation potential. Under the regulation of different growth factors, they can differentiate into different tissue cell types derived from two germ layers, such as fat. Cells, osteoblasts, hepatocytes, cardiomyocytes, glial cells, etc. HAMSCs do not express major histocompatibility class II antigens, but slightly express major histocompatibility class I antigens, with low immunogenicity and transplant rejection It has a small effect and has immunomodulatory effects, which is of great significance in stem cell therapy, tissue engineering therapy and regenerative medicine research applications.

现有羊膜间充质干细胞提取方法，将羊膜从绒毛膜上分离，用不同浓度的胰酶，中性蛋白酶或其他消化酶进行不同时间的消化将hAEC从基底膜上消化下来，然后用胶原酶对多为hAMSC进行消化分离，过程较为复杂，操作时间长，且得率不高。The existing amniotic membrane mesenchymal stem cell extraction method is to separate the amniotic membrane from the chorion, digest the hAEC from the basement membrane with different concentrations of trypsin, neutral protease or other digestive enzymes for different times, and then use collagenase. Digestion and separation of mostly hAMSCs is complicated, the operation time is long, and the yield is not high.

发明内容SUMMARY OF THE INVENTION

为了解决现有技术中存在的上述问题，本发明提供一种羊膜间充质干细胞的培养及冻存方法。所述培养方法在羊膜间充质干细胞分离阶段，通过采用特殊的混合酶消化液体系进行消化，能够更为有效的从羊膜组织中分离羊膜间充质干细胞，进而显著提高P0代细胞得率，本发明采用一步消化法，相对传统的两步消化，操作更便捷，培养得到的羊膜间充质干细胞活性好，得率高、纯度高，重复性好。In order to solve the above-mentioned problems in the prior art, the present invention provides a culture and cryopreservation method of amniotic mesenchymal stem cells. In the culture method, in the separation stage of amniotic membrane mesenchymal stem cells, by adopting a special mixed enzyme digestion liquid system for digestion, the amniotic membrane mesenchymal stem cells can be more effectively separated from the amniotic membrane tissue, thereby significantly improving the yield of P0 generation cells, Compared with the traditional two-step digestion, the invention adopts a one-step digestion method, and the operation is more convenient, and the cultured amniotic membrane mesenchymal stem cells have good activity, high yield, high purity and good repeatability.

本发明的技术方案为：The technical scheme of the present invention is:

一种羊膜间充质干细胞的培养方法，包括如下步骤：A method for culturing amniotic membrane mesenchymal stem cells, comprising the following steps:

(1)羊膜组织分离：取胎盘组织，经浸泡、洗涤后，分离得到羊膜组织；(1) Separation of amniotic membrane tissue: take placental tissue, soak and wash, and then separate the amniotic membrane tissue;

(2)羊膜间充质干细胞分离：向羊膜组织中加入混合酶消化液进行消化，过滤收集滤液，将滤液离心后弃上清，得到沉淀物；(2) Isolation of amniotic membrane mesenchymal stem cells: adding mixed enzyme digestion solution to the amniotic membrane tissue for digestion, filtering and collecting the filtrate, centrifuging the filtrate and discarding the supernatant to obtain a precipitate;

(3)P0代羊膜间充质干细胞培养：向步骤(2)所述沉淀物中加入无血清培养基进行重悬，得到细胞悬液，经接种、培养后，加入胰蛋白酶消化，根据上皮细胞和间质细胞对胰蛋白酶的耐受性不同,在消化细胞时,常是间质细胞先脱壁,而上皮细胞要消化相当长时间才脱壁。细胞消化2-3min时，间充质干细胞即可消化下来,收集消化下来的细胞离心，弃上清，所得沉淀物即为P0代羊膜间充质干细胞；(3) P0 generation amniotic mesenchymal stem cell culture: add serum-free medium to the sediment described in step (2) for resuspending to obtain a cell suspension, after inoculation and culture, add trypsin digestion, according to epithelial cells Unlike mesenchymal cells, which are resistant to trypsin, when cells are digested, mesenchymal cells are often detached first, while epithelial cells need to be digested for a long time before they detach. When the cells are digested for 2-3 minutes, the mesenchymal stem cells can be digested, and the digested cells are collected and centrifuged, and the supernatant is discarded. The resulting precipitate is the P0 generation amniotic mesenchymal stem cells;

(4)扩增培养：将步骤(3)所述P0代羊膜间充质干细胞分种至新的无血清培养基中进行传代培养，通过2至3次传代，传至P3代时进行收获，得到纯化的羊膜间充质干细胞；(4) Amplification culture: the P0 generation amniotic membrane mesenchymal stem cells described in step (3) are separated into a new serum-free medium for subculture, and after 2 to 3 passages, they are harvested when they are passed to the P3 generation, Obtain purified amniotic mesenchymal stem cells;

所述混合酶消化液按如下方法制备：将中性蛋白酶、脱氧核糖核酸酶I以及胶原酶IV溶于DME/F12中，即得；所述混合酶消化液中各组分的终浓度为：1.5-2U/mL中性蛋白酶、0.5mg/mL脱氧核糖核酸酶I、1mg/mL胶原酶IV。The mixed enzyme digestion solution is prepared as follows: neutral protease, DNase I and collagenase IV are dissolved in DME/F12, and the final concentration of each component in the mixed enzyme digestion solution is: 1.5-2 U/mL neutral protease, 0.5 mg/mL deoxyribonuclease I, 1 mg/mL collagenase IV.

优选的，步骤(1)中，先用PBS缓冲液浸泡2-3min，再用75％酒精浸泡1-2min，然后用生理盐水进行所述洗涤。Preferably, in step (1), first soak in PBS buffer for 2-3 minutes, then soak in 75% alcohol for 1-2 minutes, and then perform the washing with physiological saline.

优选的，步骤(2)中，所述羊膜组织与混合酶消化液之间的体积比为1:0.8-1.2，所述消化为在37℃条件下消化90-120min；所述过滤为采用150-250目过滤网过滤，所述离心的转速为1500-2000rpm，所述离心的时间为6-10min。Preferably, in step (2), the volume ratio between the amniotic tissue and the mixed enzyme digestion solution is 1:0.8-1.2, and the digestion is performed at 37°C for 90-120 min; the filtration is performed by using 150 -250 mesh filter screen, the centrifugal speed is 1500-2000rpm, and the centrifugal time is 6-10min.

优选的，步骤(3)中，所述无血清培养基的组成为：90％a-MEM+5％血小板裂解液+10ng/ml表皮细胞生长因子+10ng/ml重组碱性成纤维细胞生长因子+2mmol/L L-谷氨酰胺，所述接种密度为1-3×104个/cm2，所述培养为在37±0.5℃，75±5％RH，5±0.5％CO2的培养箱中进行培养，进行所述培养至细胞生长达到80-90％融合；所述消化为在37℃条件下消化2-3min，进行所述消化至细胞呈圆球状；所述离心的转速为1000-1500rpm，所述离心的时间为4-6min。Preferably, in step (3), the composition of the serum-free medium is: 90% a-MEM+5% platelet lysate+10ng/ml epidermal growth factor+10ng/ml recombinant basic fibroblast growth factor +2mmol/L L-glutamine, the seeding density is 1-3×10 4 /cm 2 , the culture is at 37±0.5°C, 75±5% RH, 5±0.5% CO 2 The culture is carried out in a box, and the culture is carried out until the cell growth reaches 80-90% confluence; the digestion is carried out at 37 ° C for 2-3 min, and the digestion is carried out until the cells are spherical; the rotation speed of the centrifugation is 1000 -1500rpm, the centrifugation time is 4-6min.

优选的，步骤(4)中，所述分种率为1:3-1:6，所述传代培养为在37±0.5℃，75±5％RH，5±0.5％CO2的培养箱中进行培养。Preferably, in step (4), the seeding ratio is 1:3-1:6, and the subculture is performed in an incubator at 37±0.5°C, 75±5% RH, 5±0.5% CO 2 to cultivate.

所述方法制备得到的羊膜间充质干细胞。The amniotic membrane mesenchymal stem cells prepared by the method.

所述羊膜间充质干细胞的冻存方法，包括如下步骤：The cryopreservation method of the amniotic membrane mesenchymal stem cells comprises the following steps:

(1)向羊膜间充质干细胞中加入胰蛋白酶进行消化，完毕后离心弃去上清，加入无血清冻存液重悬细胞沉淀，调整细胞密度，得到细胞悬液；(1) Add trypsin to the amniotic membrane mesenchymal stem cells for digestion, centrifuge and discard the supernatant after completion, add serum-free cryopreservation solution to resuspend the cell pellet, adjust the cell density, and obtain a cell suspension;

(2)将细胞悬液分装至冻存管中，降温后，转移至液氮中保存。(2) Dispense the cell suspension into cryopreservation tubes, and after cooling, transfer to liquid nitrogen for storage.

优选的，步骤(1)中，所述消化为在37℃条件下消化2-3min，所述离心为在4℃，1200rpm条件下离心3-7min。Preferably, in step (1), the digestion is at 37° C. for 2-3 minutes, and the centrifugation is at 4° C. and 1200 rpm for 3-7 minutes.

优选的，步骤(1)中，所述调整至细胞密度为0.5-1.5×107个/mL。Preferably, in step (1), the cell density is adjusted to be 0.5-1.5×10 7 cells/mL.

优选的，步骤(2)中，所述降温为先由室温降温至4℃，保持20min，再降温至-20℃，保持30min，然后降温至-80℃，保持24-72h。Preferably, in step (2), the cooling is to first cool down from room temperature to 4°C, hold for 20min, then cool to -20°C, hold for 30min, and then cool to -80°C and hold for 24-72h.

本发明的有益效果为：The beneficial effects of the present invention are:

1、本发明所述羊膜间充质干细胞的培养方法，在羊膜间充质干细胞分离阶段，通过采用特殊的混合酶消化液体系(消化液中各组分终浓度：1.5-2U/mL中性蛋白酶、0.5mg/mL脱氧核糖核酸酶I以及1mg/mL胶原酶IV)进行消化，其中加入脱氧核糖核酸酶I能使粘连的细胞分散，形成单个细胞，细胞更易透过过滤网，得率更高，能够更为有效的从羊膜组织中分离羊膜间充质干细胞，进而显著提高P0代细胞得率，进一步地，本发明采用一步消化法，相对传统的两步消化，操作更便捷，缩短了消化时间，且用该方法得到的P0代细胞活率均在95％以上，本发明培养得到P3代细胞流式检测分析结果表明，CD73、CD90、CD105为阳性，表达率大于90％，CD11b、CD19、CD34、CD45、HLA-DR为阴性，表达率均小于2％，符合间充质干细胞特征，即本发明培养方法培养得到的羊膜间充质干细胞活性好，得率高、纯度高，重复性好。1. The method for culturing amniotic mesenchymal stem cells according to the present invention, in the separation stage of amniotic mesenchymal stem cells, by adopting a special mixed enzyme digestion solution system (final concentration of each component in the digestion solution: 1.5-2U/mL neutral) Protease, 0.5mg/mL deoxyribonuclease I and 1mg/mL collagenase IV) for digestion, wherein the addition of deoxyribonuclease I can disperse the adherent cells to form single cells, the cells are more likely to pass through the filter screen, and the yield is higher. It can more effectively separate the amniotic membrane mesenchymal stem cells from the amniotic membrane tissue, thereby significantly improving the yield of the P0 generation cells. Furthermore, the present invention adopts a one-step digestion method, which is more convenient to operate compared to the traditional two-step digestion, and shortens the time required for digestion. Digestion time, and the viability of the P0 generation cells obtained by this method is above 95%, the flow detection analysis results of the P3 generation cells obtained by the culture of the present invention show that CD73, CD90, CD105 are positive, the expression rate is greater than 90%, CD11b, CD19, CD34, CD45 and HLA-DR are negative, and the expression rates are all less than 2%, which is in line with the characteristics of mesenchymal stem cells, that is, the amniotic membrane mesenchymal stem cells cultured by the culture method of the present invention have good activity, high yield, high purity, and repeatability. good sex.

2、本发明所述羊膜间充质干细胞的培养方法，在P0代羊膜间充质干细胞培养阶段、扩增培养阶段均采用无血清培养基(组成为：90％a-MEM+5％血小板裂解液+10ng/ml表皮细胞生长因子+10ng/ml重组碱性成纤维细胞生长因子+2mmol/L L-谷氨酰胺)，完全避免了动物源成分的引入，减少了由血清带来病毒、真菌和支原体等微生物污染的危险,同时也降低了变异的几率。该无血清培养体系能稳定的维持人羊膜间充质干细胞正常生物学特及功能，该培养体系培养的人羊膜间充质干细胞质量稳定，可控，符合《细胞治疗产品研究与评价技术指导原则》临床级干细胞制备标准，确保了干细胞临床应用的安全性。2. The method for culturing amniotic membrane mesenchymal stem cells of the present invention adopts serum-free medium (composition: 90% a-MEM+5% platelet lysis) in both the culture stage and the expansion culture stage of P0 generation amnion mesenchymal stem cells. liquid + 10ng/ml epidermal cell growth factor + 10ng/ml recombinant basic fibroblast growth factor + 2mmol/L L-glutamine), which completely avoids the introduction of animal-derived components and reduces the introduction of viruses and fungi from serum The risk of contamination by microorganisms such as mycoplasma and mycoplasma also reduces the chance of mutation. The serum-free culture system can stably maintain the normal biological characteristics and functions of human amniotic mesenchymal stem cells. The quality of human amniotic mesenchymal stem cells cultured in this culture system is stable and controllable, and conforms to the "Technical Guidelines for Research and Evaluation of Cell Therapy Products" 》Clinical-grade stem cell preparation standards to ensure the safety of clinical application of stem cells.

附图说明Description of drawings

为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

图1：本发明实施例1得到的P3代羊膜间充质干细胞形态图(40倍放大)Figure 1: Morphological diagram of P3 generation amniotic mesenchymal stem cells obtained in Example 1 of the present invention (40 times magnification)

图2：流式细胞仪检测本发明实施例1得到的P3代羊膜间充质干细胞表面标志CD90表达结果；Figure 2: Flow cytometry to detect the expression results of the surface marker CD90 of the P3 generation amniotic mesenchymal stem cells obtained in Example 1 of the present invention;

图3：流式细胞仪检测本发明实施例1得到的P3代羊膜间充质干细胞表面标志CD105表达结果；Figure 3: Flow cytometry to detect the expression results of the surface marker CD105 of the P3 generation amniotic mesenchymal stem cells obtained in Example 1 of the present invention;

图4：流式细胞仪检测本发明实施例1得到的P3代羊膜间充质干细胞表面标志CD73表达结果；Figure 4: Flow cytometry detection of the expression results of the surface marker CD73 of the P3 generation amniotic mesenchymal stem cells obtained in Example 1 of the present invention;

图5：流式细胞仪检测本发明实施例1得到的P3代羊膜间充质干细胞阴性表型NegCKTL(包括CD11b、CD19、CD34、CD45、HLA-DR)流式检测结果；Figure 5: Flow cytometry detection results of the negative phenotype NegCKTL (including CD11b, CD19, CD34, CD45, HLA-DR) of the P3 generation amniotic mesenchymal stem cells obtained in Example 1 of the present invention;

其中1为同型对照，2为羊膜间充质干细胞表面标志CD90(图2)\CD105(图3)\CD73(图4)\NegCKTL(图5)表达结果。Among them, 1 is the isotype control, and 2 is the expression result of the surface marker CD90 (Figure 2)\CD105 (Figure 3)\CD73 (Figure 4)\NegCKTL (Figure 5) of amniotic mesenchymal stem cells.

具体实施方式Detailed ways

为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。以下实施例中，血小板裂解液购自达科为，无血清冻存液购自BI公司(货号：05-712-1E)。In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention. In the following examples, platelet lysate was purchased from Daktronics, and serum-free cryopreservation solution was purchased from BI Company (Item No.: 05-712-1E).

实施例1Example 1

本实施例提供一种羊膜间充质干细胞的培养方法，包括如下步骤：The present embodiment provides a method for culturing amniotic mesenchymal stem cells, comprising the following steps:

(1)羊膜组织分离：将取来的胎盘组织浸泡于PBS缓冲液中，取样血水为样本来源微生物检测样本，然后再将胎盘组织浸泡2min，之后用75％的酒精浸泡胎盘组织约1min，用生理盐水反复冲洗胎盘组织后，用组织镊轻轻撕下羊膜组织，浸泡于PBS中，用于羊膜间充质干细胞分离，将最后冲洗胎盘的生理盐水取样检测微生物；(1) Separation of amniotic membrane tissue: Soak the taken placental tissue in PBS buffer, sample blood water as the sample source microorganism detection sample, then soak the placental tissue for 2 minutes, then soak the placental tissue with 75% alcohol for about 1 minute, and use After repeated washing of the placental tissue with normal saline, the amniotic membrane tissue was gently torn off with tissue forceps, soaked in PBS for the separation of amniotic mesenchymal stem cells, and the physiological saline from the final washing of the placenta was sampled to detect microorganisms;

(2)羊膜间充质干细胞分离：将羊膜组织剪成1-2mm2小块，加入到50mL无菌离心管中，并加入等体积的混合酶消化液(将中性蛋白酶、脱氧核糖核酸酶I以及胶原酶IV溶于DME/F12中，即得，终浓度为：1.5U/mL中性蛋白酶、0.5mg/mL脱氧核糖核酸酶I、1mg/mL胶原酶IV)，于37℃条件下消化90min，将所有组织及消化液于200目过滤网过滤，收集滤液后，将滤液转移至50ml离心管中，以1800rpm的转速离心8min，小心吸弃上清，得到沉淀物；(2) Isolation of amniotic mesenchymal stem cells: Cut the amniotic membrane tissue into 1-2 mm pieces, add them to a 50 mL sterile centrifuge tube, and add an equal volume of mixed enzyme digestion solution (neutral protease, deoxyribonuclease I and collagenase IV were dissolved in DME/F12, and the final concentration was: 1.5U/mL neutral protease, 0.5mg/mL deoxyribonuclease I, 1mg/mL collagenase IV), at 37°C Digest for 90min, filter all the tissues and digestive solution through a 200-mesh filter, after collecting the filtrate, transfer the filtrate to a 50ml centrifuge tube, centrifuge at 1800rpm for 8min, carefully aspirate the supernatant to obtain a precipitate;

(3)P0代羊膜间充质干细胞培养：向步骤(2)所述沉淀物中加入无血清培养基(组成：90％a-MEM+5％血小板裂解液+10ng/ml表皮细胞生长因子+10ng/ml重组碱性成纤维细胞生长因子+2mmol/L L-谷氨酰胺)重悬，按1×104个/cm2的密度接种到T75培养瓶中，培养瓶中无血清培养基体积为15mL，置于37℃、75％RH、5％CO2的培养箱中进行原代细胞培养。培养48h，显微镜下观察细胞贴壁情况，为细胞更换新鲜培养基。待细胞培养至细胞生长达到80％融合时，用DPBS洗液洗两遍后，每T75培养瓶中加入2mL胰蛋白酶消化细胞，37℃消化2min，显微镜下观察细胞变圆后，将细胞从瓶底轻轻拍打下来，将细胞悬液吸入到50mL离心管中，并将每T75培养瓶中再加入10mLDPBS清洗两次，并将洗液一并加入到已收集的细胞悬液中，4℃，1200rpm，离心5min。弃上清，所得细胞沉淀即为P0代羊膜间充质干细胞，加入无血清培养基混匀细胞沉淀，取100μL计数，得到的细胞数即为P0代羊膜间充质干细胞收获数；(3) P0 generation amniotic mesenchymal stem cell culture: add serum-free medium (composition: 90% a-MEM+5% platelet lysate+10ng/ml epidermal cell growth factor+ 10ng/ml recombinant basic fibroblast growth factor + 2mmol/L L-glutamine), resuspended, inoculated into T75 culture flasks at a density of 1 ×104/ cm2 , and the volume of serum-free medium in the culture flasks For 15 mL, place in an incubator at 37°C, 75% RH, 5% CO 2 for primary cell culture. After culturing for 48 h, the adherence of the cells was observed under a microscope, and fresh medium was replaced for the cells. When the cells were cultured until the cell growth reached 80% confluence, after washing twice with DPBS wash solution, add 2 mL of trypsinized cells to each T75 culture flask, digested at 37 °C for 2 min, and observed that the cells became rounded under a microscope, and removed the cells from the flask. The bottom was gently tapped, the cell suspension was sucked into a 50mL centrifuge tube, and 10mL of DPBS was added to each T75 culture flask to wash twice, and the washes were added to the collected cell suspension at 4°C. 1200rpm, centrifuged for 5min. Discard the supernatant, the obtained cell pellet is P0 generation amniotic mesenchymal stem cells, add serum-free medium to mix the cell pellet, take 100 μL for counting, and the number of cells obtained is the harvested number of P0 generation amniotic mesenchymal stem cells;

(4)扩增培养：将获得的P0代羊膜间充质干细胞按分种率1:3接种到添加有新无血清培养基的培养瓶中培养，在每个T75培养瓶中加入15ml无血清培养基培养，置于37℃、75％RH、5％CO2培养箱中进行培养，待细胞生长达到80％融合，用DPBS洗液洗两遍后，每T75培养瓶中加入2mL胰蛋白酶消化细胞，37℃消化2min，显微镜下观察细胞变圆后，将细胞从瓶底轻轻拍打下来，将细胞悬液吸入到50mL离心管中，并向每T75培养瓶中再加入10mLDPBS清洗两次，将洗液一并加入到已收集的细胞悬液中，4℃，1200rpm，离心5min，所得细胞沉淀即为P1代羊膜间充质干细胞，同样方法将P1代羊膜间充质干细胞传至P3代，收获得到纯化的羊膜间充质干细胞。(4) Expansion culture: Inoculate the obtained P0 amniotic mesenchymal stem cells into culture flasks supplemented with new serum-free medium at a seeding rate of 1:3, and add 15 ml of serum-free culture to each T75 culture flask basal culture, placed in a 37°C, 75% RH, 5% CO 2 incubator for culture, when the cells reached 80% confluence, washed twice with DPBS wash solution, add 2 mL of trypsinized cells to each T75 culture flask , digested at 37°C for 2 min, and observed that the cells became rounded under a microscope, tap the cells from the bottom of the bottle gently, aspirate the cell suspension into a 50 mL centrifuge tube, and add 10 mL of DPBS to each T75 culture flask to wash twice. The washing solution was added to the collected cell suspension, centrifuged at 4°C, 1200 rpm for 5 min, and the obtained cell pellet was the P1 generation amnion mesenchymal stem cells. The same method was used to pass the P1 generation amniotic membrane mesenchymal stem cells to the P3 generation. The purified amniotic mesenchymal stem cells were harvested.

所述P3代羊膜间充质干细胞的冻存方法，包括如下步骤：The cryopreservation method of the P3 generation amniotic mesenchymal stem cells comprises the following steps:

(1)P3代羊膜间充质干细胞冻存前24h，换新鲜无血清培养基，使细胞一直处于对数生长期，待细胞生长至80％融合时，加入2mL胰蛋白酶消化细胞，37℃消化2min，显微镜下观察，细胞呈现圆球状态时，将细胞从瓶底轻轻拍打下来，将细胞悬液吸入到50mL离心管中，并将每T75培养瓶中再加入10mLDPBS清洗两次，将洗液一并加入到已收集的细胞悬液中，4℃，1200rpm，离心5min，弃去上清，加入无血清冻存液重悬细胞沉淀，调整细胞密度为1×107个/mL。(1) 24 hours before cryopreservation of P3 amniotic mesenchymal stem cells, change to fresh serum-free medium to keep the cells in the logarithmic growth phase. When the cells grow to 80% confluence, add 2 mL of trypsin to digest the cells and digest at 37°C 2min, observed under the microscope, when the cells are in spherical state, gently tap the cells from the bottom of the bottle, suck the cell suspension into a 50mL centrifuge tube, and add 10mL DPBS to each T75 culture flask to wash twice. The solution was added to the collected cell suspension, centrifuged at 4°C, 1200 rpm for 5 min, the supernatant was discarded, and the serum-free freezing medium was added to resuspend the cell pellet, and the cell density was adjusted to 1×10 7 cells/mL.

(2)将细胞悬液分装至冻存管中，每管1mL，先由室温降温至4℃，保持20min，再降温至-20℃，保持30min，然后降温至-80℃，24h后转移至液氮中保存。(2) Dispense the cell suspension into cryopreservation tubes, 1 mL per tube, first cool down from room temperature to 4°C, hold for 20 minutes, then cool down to -20°C, hold for 30 minutes, then cool down to -80°C, transfer after 24 hours Store in liquid nitrogen.

经鉴定，P0代羊膜间充质干细胞的细胞形态呈梭形、多角形，P0代羊膜间充质干细胞约8天接近80％融合，传代后细胞纯度提高，形态较原代均一，以平行排列生长为主，或呈漩涡状生长(见图1)；P3代羊膜间充质干细胞流式检测分析结果表明，CD73、CD90、CD105为阳性，表达率大于90％，CD11b、CD19、CD34、CD45、HLA-DR为阴性，表达率均小于2％，符合间充质干细胞特征(见图2-5)。It has been identified that the cell morphology of P0 generation amniotic mesenchymal stem cells is spindle-shaped and polygonal, and the P0 generation amniotic mesenchymal stem cells are nearly 80% confluent in about 8 days. Mainly growing, or swirling growth (see Figure 1); P3 amniotic mesenchymal stem cells flow detection analysis results show that CD73, CD90, CD105 are positive, the expression rate is greater than 90%, CD11b, CD19, CD34, CD45 , HLA-DR were negative, the expression rate was less than 2%, in line with the characteristics of mesenchymal stem cells (see Figure 2-5).

实施例2Example 2

本实施例提供一种羊膜间充质干细胞的培养方法，包括如下步骤：The present embodiment provides a method for culturing amniotic mesenchymal stem cells, comprising the following steps:

(1)羊膜组织分离：将取来的胎盘组织浸泡于PBS缓冲液中，取样血水为样本来源微生物检测样本，然后再将胎盘组织浸泡3min，之后用75％的酒精浸泡胎盘组织约2min，用生理盐水反复冲洗胎盘组织后，用组织镊轻轻撕下羊膜组织，浸泡于PBS中，用于羊膜间充质干细胞分离，将最后冲洗胎盘的生理盐水取样检测微生物；(1) Separation of amniotic membrane tissue: Soak the placental tissue in PBS buffer, sample blood water as the sample source for microbial detection, then soak the placental tissue for 3 minutes, then soak the placental tissue with 75% alcohol for about 2 minutes, and use After repeated washing of the placental tissue with normal saline, the amniotic membrane tissue was gently torn off with tissue forceps, soaked in PBS for the separation of amniotic mesenchymal stem cells, and the physiological saline from the final washing of the placenta was sampled to detect microorganisms;

(2)羊膜间充质干细胞分离：将羊膜组织剪成1-2mm2小块，加入到50mL无菌离心管中，并加入0.8倍羊膜组织体积的混合酶消化液(将中性蛋白酶、脱氧核糖核酸酶I以及胶原酶IV溶于DME/F12中，即得，终浓度为：2U/mL中性蛋白酶、0.5mg/mL脱氧核糖核酸酶I、1mg/mL胶原酶IV)，于37℃条件下消化120min，将所有组织及消化液于150目过滤网过滤，收集滤液后，将滤液转移至50ml离心管中，以1500rpm的转速离心10min，小心吸弃上清，得到沉淀物；(2) Isolation of amniotic mesenchymal stem cells: Cut the amniotic tissue into 1-2 mm pieces, add them to a 50 mL sterile centrifuge tube, and add 0.8 times the volume of the amniotic tissue volume of mixed enzyme digestion solution (neutral protease, deoxygenation Ribonuclease I and collagenase IV were dissolved in DME/F12, and the final concentration was: 2U/mL neutral protease, 0.5mg/mL deoxyribonuclease I, 1mg/mL collagenase IV), at 37°C Digest for 120 min under conditions, filter all tissues and digestive solution through a 150-mesh filter, after collecting the filtrate, transfer the filtrate to a 50 ml centrifuge tube, centrifuge at 1500 rpm for 10 min, carefully aspirate and discard the supernatant to obtain a precipitate;

(3)P0代羊膜间充质干细胞培养：向步骤(2)所述沉淀物中加入无血清培养基(组成：90％a-MEM+5％血小板裂解液+10ng/ml表皮细胞生长因子+10ng/ml重组碱性成纤维细胞生长因子+2mmol/L L-谷氨酰胺)重悬，按3×104个/cm2的密度接种到T75培养瓶中，培养瓶中无血清培养基体积为15mL，置于36.5℃、70％RH、4.5％CO2的培养箱中进行原代细胞培养。培养48h，显微镜下观察细胞贴壁情况，为细胞更换新鲜培养基。待细胞培养至细胞生长达到90％融合时，用DPBS洗液洗两遍后，每T75培养瓶中加入2mL胰蛋白酶消化细胞，37℃消化3min，显微镜下观察细胞变圆后，将细胞从瓶底轻轻拍打下来，将细胞悬液吸入到50mL离心管中，并将每T75培养瓶中再加入10mLDPBS清洗两次，并将洗液一并加入到已收集的细胞悬液中，4℃，1000rpm，离心6min。弃上清，所得细胞沉淀即为P0代羊膜间充质干细胞，加入无血清培养基混匀细胞沉淀，取100μL计数，得到的细胞数即为P0代细胞收获数；(3) P0 generation amniotic mesenchymal stem cell culture: add serum-free medium (composition: 90% a-MEM+5% platelet lysate+10ng/ml epidermal cell growth factor+ 10ng/ml recombinant basic fibroblast growth factor + 2mmol/L L-glutamine), resuspended, inoculated into T75 culture flasks at a density of 3 ×104/ cm2 , and the volume of serum-free medium in the culture flasks 15 mL, placed in an incubator at 36.5 °C, 70% RH, 4.5% CO 2 for primary cell culture. After culturing for 48 h, the adherence of the cells was observed under a microscope, and fresh medium was replaced for the cells. When the cells were cultured until the cell growth reached 90% confluence, after washing twice with DPBS wash solution, 2 mL of trypsinized cells were added to each T75 culture flask, and the cells were digested at 37°C for 3 min. The bottom was gently tapped, the cell suspension was sucked into a 50mL centrifuge tube, and 10mL of DPBS was added to each T75 culture flask to wash twice, and the washes were added to the collected cell suspension at 4°C. 1000rpm, centrifugation for 6min. Discard the supernatant, the obtained cell pellet is P0 generation amniotic mesenchymal stem cells, add serum-free medium to mix the cell pellet, take 100 μL for counting, and the number of cells obtained is the harvested number of P0 generation cells;

(4)扩增培养：将获得的P0代羊膜间充质干细胞按分种率1:6接种到添加有新无血清培养基的培养瓶中培养，在每个T75培养瓶中加入15ml无血清培养基培养，置于36.5℃、70％RH、4.5％CO2培养箱中进行培养，待细胞生长达到90％融合，用DPBS洗液洗两遍后，每T75培养瓶中加入2mL胰蛋白酶消化细胞，37℃消化3min，显微镜下观察细胞变圆后，将细胞从瓶底轻轻拍打下来，将细胞悬液吸入到50mL离心管中，并向每T75培养瓶中再加入10mLDPBS清洗两次，将洗液一并加入到已收集的细胞悬液中，4℃，1200rpm，离心4min，所得细胞沉淀即为P1代羊膜间充质干细胞，同样方法将P1代羊膜间充质干细胞传至P3代，收获得到纯化的羊膜间充质干细胞。(4) Expansion culture: Inoculate the obtained P0 amniotic mesenchymal stem cells into culture flasks supplemented with new serum-free medium at a seeding rate of 1:6, and add 15ml of serum-free culture to each T75 culture flask basal culture, placed in a 36.5°C, 70% RH, 4.5% CO 2 incubator for culturing, when the cells reached 90% confluence, washed twice with DPBS wash solution, add 2 mL of trypsinized cells to each T75 culture flask , digested at 37°C for 3 min, and observed that the cells became rounded under a microscope, tap the cells from the bottom of the bottle gently, suck the cell suspension into a 50 mL centrifuge tube, and add 10 mL of DPBS to each T75 culture flask to wash twice. The washing solution was added to the collected cell suspension, centrifuged at 4°C, 1200 rpm for 4 minutes, and the obtained cell pellet was the P1 generation amnion mesenchymal stem cells. The same method was used to transfer the P1 generation amniotic membrane mesenchymal stem cells to the P3 generation. The purified amniotic mesenchymal stem cells were harvested.

所述P3代羊膜间充质干细胞的冻存方法，包括如下步骤：The cryopreservation method of the P3 generation amniotic mesenchymal stem cells comprises the following steps:

(1)P3代羊膜间充质干细胞冻存前24h，换新鲜无血清培养基，使细胞一直处于对数生长期，待细胞生长至90％融合时，加入2mL胰蛋白酶消化细胞，37℃消化3min，显微镜下观察，细胞呈现圆球状态时，将细胞从瓶底轻轻拍打下来，将细胞悬液吸入到50mL离心管中，并将每T75培养瓶中再加入10mLDPBS清洗两次，将洗液一并加入到已收集的细胞悬液中，4℃，1200rpm，离心3min，弃去上清，加入无血清冻存液重悬细胞沉淀，调整细胞密度为0.5×107个/mL。(1) 24 hours before cryopreservation of P3 amniotic mesenchymal stem cells, change to fresh serum-free medium to keep the cells in the logarithmic growth phase. When the cells grow to 90% confluence, add 2 mL of trypsin to digest the cells and digest at 37°C 3min, observed under the microscope, when the cells appeared spherical state, gently tap the cells from the bottom of the bottle, suck the cell suspension into a 50mL centrifuge tube, and add 10mL DPBS to each T75 culture flask to wash twice. Add the collected cell suspension to the collected cell suspension, centrifuge for 3 min at 4°C, 1200 rpm, discard the supernatant, add serum-free freezing medium to resuspend the cell pellet, and adjust the cell density to 0.5×10 7 cells/mL.

(2)将细胞悬液分装至冻存管中，每管1mL，先由室温降温至4℃，保持20min，再降温至-20℃，保持30min，然后降温至-80℃，72h后转移至液氮中保存。(2) Dispense the cell suspension into cryopreservation tubes, 1 mL per tube, first cool down from room temperature to 4°C, hold for 20 minutes, then cool down to -20°C, hold for 30 minutes, then cool down to -80°C, transfer after 72 hours Store in liquid nitrogen.

经鉴定，P3代羊膜间充质干细胞流式检测分析结果表明，CD73、CD90、CD105为阳性，表达率大于90％，CD11b、CD19、CD34、CD45、HLA-DR为阴性，表达率均小于2％，符合间充质干细胞特征。After identification, the results of flow detection analysis of P3 generation amniotic mesenchymal stem cells showed that CD73, CD90, CD105 were positive, and the expression rate was greater than 90%, and CD11b, CD19, CD34, CD45, and HLA-DR were negative, and the expression rates were all less than 2. %, consistent with the characteristics of mesenchymal stem cells.

实施例3Example 3

本实施例提供一种羊膜间充质干细胞的培养方法，包括如下步骤：The present embodiment provides a method for culturing amniotic mesenchymal stem cells, comprising the following steps:

(1)羊膜组织分离：将取来的胎盘组织浸泡于PBS缓冲液中，取样血水为样本来源微生物检测样本，然后再将胎盘组织浸泡3min，之后用75％的酒精浸泡胎盘组织约2min，用生理盐水反复冲洗胎盘组织后，用组织镊轻轻撕下羊膜组织，浸泡于PBS中，用于羊膜间充质干细胞分离，将最后冲洗胎盘的生理盐水取样检测微生物；(1) Separation of amniotic membrane tissue: Soak the placental tissue in PBS buffer, sample blood water as the sample source for microbial detection, then soak the placental tissue for 3 minutes, then soak the placental tissue with 75% alcohol for about 2 minutes, and use After repeated washing of the placental tissue with normal saline, the amniotic membrane tissue was gently torn off with tissue forceps, soaked in PBS for the separation of amniotic mesenchymal stem cells, and the physiological saline from the final washing of the placenta was sampled to detect microorganisms;

(2)羊膜间充质干细胞分离：将羊膜组织剪成1-2mm2小块，加入到50mL无菌离心管中，并加入1.2倍羊膜组织体积的混合酶消化液(将中性蛋白酶、脱氧核糖核酸酶I以及胶原酶IV溶于DME/F12中，即得，终浓度为：1.5U/mL中性蛋白酶、0.5mg/mL脱氧核糖核酸酶I、1mg/mL胶原酶IV)，于37℃条件下消化100min，将所有组织及消化液于250目过滤网过滤，收集滤液后，将滤液转移至50ml离心管中，以2000rpm的转速离心6min，小心吸弃上清，得到沉淀物；(2) Isolation of amniotic membrane mesenchymal stem cells: cut the amniotic membrane tissue into 1-2 mm pieces, add them to a 50 mL sterile centrifuge tube, and add 1.2 times the volume of amniotic membrane tissue with mixed enzyme digestion solution (neutral protease, deoxygenation Ribonuclease I and collagenase IV were dissolved in DME/F12, and the final concentration was: 1.5U/mL neutral protease, 0.5mg/mL deoxyribonuclease I, 1mg/mL collagenase IV), at 37 Digest at ℃ for 100 min, filter all tissues and digestion solution through a 250-mesh filter, after collecting the filtrate, transfer the filtrate to a 50 ml centrifuge tube, centrifuge at 2000 rpm for 6 min, carefully aspirate and discard the supernatant to obtain a precipitate;

(3)P0代羊膜间充质干细胞培养：向步骤(2)所述沉淀物中加入无血清培养基(组成：90％a-MEM+5％血小板裂解液+10ng/ml表皮细胞生长因子+10ng/ml重组碱性成纤维细胞生长因子+2mmol/L L-谷氨酰胺)重悬，按2×104个/cm2的密度接种到T75培养瓶中，培养瓶中无血清培养基体积为15mL，置于37.5℃、80％RH、5.5％CO2的培养箱中进行原代细胞培养。培养48h，显微镜下观察细胞贴壁情况，为细胞更换新鲜培养基。待细胞培养至细胞生长达到80％融合时，用DPBS洗液洗两遍后，每T75培养瓶中加入2mL胰蛋白酶消化细胞，37℃消化3min，显微镜下观察细胞变圆后，将细胞从瓶底轻轻拍打下来，将细胞悬液吸入到50mL离心管中，并将每T75培养瓶中再加入10mLDPBS清洗两次，并将洗液一并加入到已收集的细胞悬液中，4℃，1500rpm，离心4min。弃上清，所得细胞沉淀即为P0代羊膜间充质干细胞，加入无血清培养基混匀细胞沉淀，取100μL计数，得到的细胞数即为P0代细胞收获数；(3) P0 generation amniotic mesenchymal stem cell culture: add serum-free medium (composition: 90% a-MEM+5% platelet lysate+10ng/ml epidermal cell growth factor+ 10ng/ml recombinant basic fibroblast growth factor + 2mmol/L L-glutamine), resuspended, inoculated into T75 culture flasks at a density of 2 ×104/ cm2 , and the volume of serum-free medium in the culture flasks 15 mL, placed in an incubator at 37.5 °C, 80% RH, 5.5% CO 2 for primary cell culture. After culturing for 48 h, the adherence of the cells was observed under a microscope, and fresh medium was replaced for the cells. When the cells were cultured until the cell growth reached 80% confluence, after washing twice with DPBS wash solution, 2 mL of trypsinized cells were added to each T75 culture flask, and the cells were digested at 37 °C for 3 min. The bottom was gently tapped, the cell suspension was sucked into a 50mL centrifuge tube, and 10mL of DPBS was added to each T75 culture flask to wash twice, and the washes were added to the collected cell suspension at 4°C. 1500rpm, centrifuged for 4min. Discard the supernatant, the obtained cell pellet is P0 generation amniotic mesenchymal stem cells, add serum-free medium to mix the cell pellet, take 100 μL for counting, and the number of cells obtained is the harvested number of P0 generation cells;

(4)扩增培养：将获得的P0代羊膜间充质干细胞按分种率1:3接种到添加有新无血清培养基的培养瓶中培养，在每个T75培养瓶中加入15ml无血清培养基培养，置于37.5℃、80％RH、5.5％CO2培养箱中进行培养，待细胞生长达到80％融合，用DPBS洗液洗两遍后，每T75培养瓶中加入2mL胰蛋白酶消化细胞，37℃消化3min，显微镜下观察细胞变圆后，将细胞从瓶底轻轻拍打下来，将细胞悬液吸入到50mL离心管中，并向每T75培养瓶中再加入10mLDPBS清洗两次，将洗液一并加入到已收集的细胞悬液中，4℃，1200rpm，离心6min，所得细胞沉淀即为P1代羊膜间充质干细胞，同样方法将P1代羊膜间充质干细胞传至P3代，收获得到纯化的羊膜间充质干细胞。(4) Expansion culture: Inoculate the obtained P0 amniotic mesenchymal stem cells into culture flasks supplemented with new serum-free medium at a seeding rate of 1:3, and add 15 ml of serum-free culture to each T75 culture flask basal culture, placed in a 37.5°C, 80% RH, 5.5% CO 2 incubator for culture, when the cells reached 80% confluence, washed twice with DPBS wash solution, add 2 mL of trypsinized cells to each T75 culture flask , digested at 37°C for 3 min, and observed that the cells became rounded under a microscope, tap the cells from the bottom of the bottle gently, suck the cell suspension into a 50 mL centrifuge tube, and add 10 mL of DPBS to each T75 culture flask to wash twice. The washing solution was added to the collected cell suspension, centrifuged at 4°C, 1200 rpm for 6 min, and the obtained cell pellet was the P1 generation amnion mesenchymal stem cells. The same method was used to transfer the P1 generation amniotic membrane mesenchymal stem cells to the P3 generation, The purified amniotic mesenchymal stem cells were harvested.

所述P3代羊膜间充质干细胞的冻存方法，包括如下步骤：The cryopreservation method of the P3 generation amniotic mesenchymal stem cells comprises the following steps:

(1)P3代羊膜间充质干细胞冻存前24h，换新鲜无血清培养基，使细胞一直处于对数生长期，待细胞生长至80％融合时，加入2mL胰蛋白酶消化细胞，37℃消化3min，显微镜下观察，细胞呈现圆球状态时，将细胞从瓶底轻轻拍打下来，将细胞悬液吸入到50mL离心管中，并将每T75培养瓶中再加入10mLDPBS清洗两次，将洗液一并加入到已收集的细胞悬液中，4℃，1200rpm，离心7min，弃去上清，加入无血清冻存液重悬细胞沉淀，调整细胞密度为1.5×107个/mL。(1) 24 hours before cryopreservation of P3 amniotic mesenchymal stem cells, change to fresh serum-free medium to keep the cells in the logarithmic growth phase. When the cells grow to 80% confluence, add 2 mL of trypsin to digest the cells and digest at 37°C 3min, observed under the microscope, when the cells appeared spherical state, gently tap the cells from the bottom of the bottle, suck the cell suspension into a 50mL centrifuge tube, and add 10mL DPBS to each T75 culture flask to wash twice. The solution was added to the collected cell suspension, centrifuged at 4°C, 1200 rpm for 7 min, the supernatant was discarded, and the cell pellet was resuspended by adding serum-free freezing medium, and the cell density was adjusted to 1.5×10 7 cells/mL.

(2)将细胞悬液分装至冻存管中，每管1mL，先由室温降温至4℃，保持20min，再降温至-20℃，保持30min，然后降温至-80℃，48h后转移至液氮中保存。(2) Dispense the cell suspension into cryopreservation tubes, 1 mL per tube, first cool down from room temperature to 4°C, hold for 20 minutes, then cool down to -20°C, hold for 30 minutes, then cool down to -80°C, transfer after 48 hours Store in liquid nitrogen.

经鉴定，P3代羊膜间充质干细胞流式检测分析结果表明，CD73、CD90、CD105为阳性，表达率大于90％，CD11b、CD19、CD34、CD45、HLA-DR为阴性，表达率均小于2％，符合间充质干细胞特征。After identification, the results of flow detection analysis of P3 generation amniotic mesenchymal stem cells showed that CD73, CD90, CD105 were positive, and the expression rate was greater than 90%, and CD11b, CD19, CD34, CD45, and HLA-DR were negative, and the expression rates were all less than 2. %, consistent with the characteristics of mesenchymal stem cells.

对比例1Comparative Example 1

与实施例1的区别在于混合酶消化液的组成不同，具体的，本对比例的混合酶消化液的组成为：1.5-2U/mL中性蛋白酶、0.5mg/mL透明质酸酶以及1mg/mL胶原酶IV。The difference from Example 1 is that the composition of the mixed enzyme digestion solution is different. Specifically, the composition of the mixed enzyme digestion solution of this comparative example is: 1.5-2U/mL neutral protease, 0.5mg/mL hyaluronidase and 1mg/mL hyaluronidase. mL of collagenase IV.

对比例2Comparative Example 2

与实施例1的区别在于去掉混合酶消化液中的脱氧核糖核酸酶I。The difference from Example 1 is that deoxyribonuclease I in the mixed enzyme digestion solution is removed.

表1 P0代羊膜间充质干细胞收获数量检测结果Table 1 The results of the number of harvested amniotic mesenchymal stem cells in P0 generation

Figure BDA0002361421250000111

Figure BDA0002361421250000111

结果表明，采用实施例1培养方法收获的P0代细胞最多，其次为实施例3、实施例2、对比例1、对比例2；对实施例1收获的羊膜间充质干细胞进行了鉴定，结果显示，P0代细胞形态呈梭形、多角形，约8天接近80％融合，传代后细胞纯度提高，形态较原代均一，以平行排列生长为主，或呈漩涡状生长(见图1)，P3代细胞流式检测分析结果表明，CD73、CD90、CD105为阳性，表达率大于90％，CD11b、CD19、CD34、CD45、HLA-DR为阴性，表达率均小于2％，符合间充质干细胞特征(见图2-5)。进一步分析认为，本发明混合酶消化液所采用的混合消化酶体系能够更为有效的从羊膜组织中分离羊膜间充质干细胞，进而显著提高P0代细胞得率，且本发明收获的P3代羊膜间充质干细胞活性好，纯度高。The results showed that the cells of the P0 generation harvested by the culture method of Example 1 were the most, followed by Example 3, Example 2, Comparative Example 1, and Comparative Example 2; the amniotic membrane mesenchymal stem cells harvested in Example 1 were identified, and the results It was shown that the cells of the P0 generation were spindle-shaped and polygonal in shape, and nearly 80% confluent in about 8 days. After passage, the purity of the cells was improved, and the morphology was more uniform than that of the primary generation. The growth was mainly parallel or swirling (see Figure 1). , P3 generation cells flow detection analysis results show that CD73, CD90, CD105 are positive, the expression rate is greater than 90%, CD11b, CD19, CD34, CD45, HLA-DR are negative, the expression rate is less than 2%, consistent with mesenchymal Stem cell characteristics (see Figures 2-5). Further analysis believes that the mixed digestive enzyme system adopted by the mixed enzyme digestion solution of the present invention can more effectively separate the amniotic membrane mesenchymal stem cells from the amniotic membrane tissue, thereby significantly improving the yield of the P0 generation cells, and the P3 generation amniotic membrane harvested by the present invention. Mesenchymal stem cells have good activity and high purity.

以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited to this. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention. should be included within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (5)

1.一种羊膜间充质干细胞的培养方法，其特征在于，包括如下步骤：1. a culture method of amniotic membrane mesenchymal stem cell, is characterized in that, comprises the steps: （1）羊膜组织分离：取胎盘组织，经浸泡、洗涤后，分离得到羊膜组织；(1) Separation of amniotic membrane tissue: take placental tissue, soak and wash, and then separate the amniotic membrane tissue; （2）羊膜间充质干细胞分离：向羊膜组织中加入混合酶消化液进行消化，过滤收集滤液，将滤液离心后弃上清，得到沉淀物；所述羊膜组织与混合酶消化液之间的体积比为1:0.8-1.2，所述消化为在37℃条件下消化90-120min；(2) Isolation of amniotic membrane mesenchymal stem cells: adding mixed enzyme digestion solution to the amniotic membrane tissue for digestion, filtering and collecting the filtrate, centrifuging the filtrate and discarding the supernatant to obtain a precipitate; The volume ratio is 1:0.8-1.2, and the digestion is 90-120min at 37°C; （3）P0代羊膜间充质干细胞培养：向步骤（2）所述沉淀物中加入无血清培养基进行重悬，得到细胞悬液，经接种、无血清培养基培养后，加入胰蛋白酶消化，然后离心，弃上清，所得沉淀物即为P0代羊膜间充质干细胞；所述无血清培养基的组成为：90%a-MEM +5%血小板裂解液+10ng/ml表皮细胞生长因子+10ng/ml重组碱性成纤维细胞生长因子+2mmol/L L-谷氨酰胺；(3) P0 generation amniotic mesenchymal stem cell culture: add serum-free medium to the sediment described in step (2) for resuspending to obtain a cell suspension. After inoculation and culture in serum-free medium, trypsin digestion is added. , then centrifuged, the supernatant was discarded, and the obtained precipitate was P0 amniotic mesenchymal stem cells; the serum-free medium was composed of: 90% a-MEM + 5% platelet lysate + 10 ng/ml epidermal cell growth factor +10ng/ml recombinant basic fibroblast growth factor+2mmol/L L-glutamine; （4）扩增培养：将步骤（3）所述P0代羊膜间充质干细胞分种至新的无血清培养基中进行传代培养，传至P3代时进行收获，得到纯化的羊膜间充质干细胞；(4) Expansion culture: the P0 generation amniotic mesenchymal stem cells described in step (3) are separated into a new serum-free medium for subculture, and harvested when they are passed to the P3 generation to obtain purified amniotic mesenchymal stem cells stem cell; 所述混合酶消化液按如下方法制备：将中性蛋白酶、脱氧核糖核酸酶I以及胶原酶IV溶于DME/F12中，即得；所述混合酶消化液中各组分的终浓度为：1.5-2U/mL中性蛋白酶、0.5mg/mL脱氧核糖核酸酶I、1mg/mL胶原酶IV。The mixed enzyme digestion solution is prepared as follows: neutral protease, DNase I and collagenase IV are dissolved in DME/F12, and the final concentration of each component in the mixed enzyme digestion solution is: 1.5-2 U/mL neutral protease, 0.5 mg/mL deoxyribonuclease I, 1 mg/mL collagenase IV. 2.根据权利要求1所述的羊膜间充质干细胞的培养方法，其特征在于，步骤（1）中，先用PBS缓冲液浸泡2-3min，再用75%酒精浸泡1-2min，然后用生理盐水进行所述洗涤。2. The method for culturing amniotic mesenchymal stem cells according to claim 1, wherein in step (1), first soak in PBS buffer for 2-3min, then soak in 75% alcohol for 1-2min, and then use Physiological saline was used for the washing. 3.根据权利要求1所述的羊膜间充质干细胞的培养方法，其特征在于，步骤（2）中，所述过滤为采用150-250目过滤网过滤，所述离心的转速为1500-2000rpm，所述离心的时间为6-10min。3 . The method for culturing amniotic mesenchymal stem cells according to claim 1 , wherein in step (2), the filtration is by using a 150-250 mesh filter screen, and the rotation speed of the centrifugation is 1500-2000 rpm , the centrifugation time is 6-10min. 4.根据权利要求1所述的羊膜间充质干细胞的培养方法，其特征在于，步骤（3）中，所述接种密度为1-3×104个/cm2，所述培养为在37±0.5℃，75±5%RH，5±0.5%CO2的培养箱中进行培养，进行所述培养至细胞生长达到80-90%融合；所述消化为在37℃条件下消化2-3min，进行所述消化至细胞呈圆球状；所述离心的转速为1000-1500rpm，所述离心的时间为4-6min。4 . The method for culturing amniotic mesenchymal stem cells according to claim 1 , wherein in step (3), the seeding density is 1-3×10 4 cells/cm 2 , and the culturing is performed at 37 . Culture in an incubator at ±0.5°C, 75±5% RH, 5±0.5% CO 2 , and carry out the culture until the cells grow to reach 80-90% confluence; the digestion is at 37°C for 2-3min , carry out the digestion until the cells are spherical; the rotating speed of the centrifugation is 1000-1500rpm, and the time of the centrifugation is 4-6min. 5.根据权利要求1所述的羊膜间充质干细胞的培养方法，其特征在于，步骤（4）中，所述分种率为1:3-1:6，所述传代培养为在37±0.5℃，75±5%RH，5±0.5%CO2的培养箱中进行培养。5 . The method for culturing amniotic mesenchymal stem cells according to claim 1 , wherein in step (4), the seeding ratio is 1:3-1:6, and the subculture is 37±1:6. 6 . Culture in an incubator at 0.5 °C, 75 ± 5% RH, 5 ± 0.5% CO 2 .

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