WO2021104360A1 - 一种提高人多功能干细胞体外悬浮培养的效率的方法 - Google Patents

一种提高人多功能干细胞体外悬浮培养的效率的方法 Download PDF

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WO2021104360A1
WO2021104360A1 PCT/CN2020/131726 CN2020131726W WO2021104360A1 WO 2021104360 A1 WO2021104360 A1 WO 2021104360A1 CN 2020131726 W CN2020131726 W CN 2020131726W WO 2021104360 A1 WO2021104360 A1 WO 2021104360A1
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段玉友
唐湘莲
吴海滨
陈洪林
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华南理工大学
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Definitions

  • the invention belongs to the technical field of cell culture, and specifically relates to a method for improving the efficiency of in vitro suspension culture of human pluripotent stem cells.
  • Human embryonic stem cells hESC
  • human induced pluripotent stem cells hPSC
  • 2D two-dimensional culture technology that uses plastic culture plates and heterogeneous culture media for cell expansion
  • 3D three-dimensional environment
  • the strategy of inoculating a single cell in a bioreactor to expand in the form of cell aggregates has the advantages of cost-saving and easy recovery of cells compared with the microcarrier culture strategy, but this strategy results in a larger size and Agglomerates of uneven size. Due to the large diameter of large aggregates (>300 ⁇ m), nutrients and oxygen are difficult to diffuse to the center of the aggregates, leading to cell differentiation or death.
  • Aggregate size control is a key parameter of the strategy of culturing cells in aggregate mode.
  • the size of aggregates can be controlled by physical methods such as controlling shear force and chemical methods such as adding small molecules to obtain a more uniform cell aggregate product.
  • Polysulfate compounds are widely used in the biopharmaceutical industry. They can not only reduce cell aggregation by regulating the charge on the cell surface, but also have an anti-apoptotic effect on cells. At the same time, it has been reported in the literature that dextran sulfate (DS) can reduce the cell aggregation of hPSCs without reducing their pluripotency. Lipsitz YY et al. [1Lipsitz YY, Tonge PD, Zandstra PW.
  • the use of DS can reduce cell aggregation by adjusting the charge on the cell surface, which is conducive to obtaining high-quality small cell aggregates that are uniform in size and beneficial to recovery, the cell expansion ratio has not been increased, that is, the number of cells is still not up to the requirement.
  • the present invention uses DS (dextran sulfate) in combination with PVA to improve the efficiency of in vitro suspension culture of stem cells, control the size of cell aggregates and increase cell expansion in the expansion strategy in the form of cell aggregates Multiplier, the pluripotency of the recovered cells can be maintained to meet the needs of in vitro expansion.
  • DS extract sulfate
  • the combined use of PVA and DS can not only obtain uniform size aggregates, but also achieve higher cell numbers to meet clinical needs.
  • the purpose of the present invention is to provide a method for improving the efficiency of in vitro suspension culture of human pluripotent stem cells.
  • the use of PVA in the present invention can promote the proliferation of hPSCs. Further, the combined use of DS (dextran sulfate) and PVA can not only obtain aggregates of uniform size, but also achieve a higher number of cells and increase stem cells. The efficiency of in vitro suspension culture.
  • the method of the present invention can be used for large-scale suspension culture of hPSC in vitro.
  • a method for improving the efficiency of in vitro suspension culture of human multifunctional stem cells including the following steps: using human multifunctional stem cells in a medium containing PVA (polyvinyl alcohol) or DS (dextran sulfate) and PVA (polyvinyl alcohol) Carry out three-dimensional training.
  • PVA polyvinyl alcohol
  • DS extract sulfate
  • PVA polyvinyl alcohol
  • the concentration of the DS (dextran sulfate) in the culture medium is 10-100 ug/ml, and its molecular weight is 40,000 kDA (D40);
  • the concentration of the PVA in the culture medium is 0.1-1 mg/ml.
  • the concentration of DS (dextran sulfate) in the medium is preferably 100ug/ml, and PVA in the medium The concentration in is preferably 1 mg/ml.
  • the medium is a serum-free complete medium, preferably mTeSR1 medium.
  • the medium was changed with PVA-containing medium, and the medium was changed once a day at 60%.
  • the method for improving the efficiency of in vitro suspension culture of human pluripotent stem cells specifically includes the following steps:
  • hPSC human pluripotent stem cells
  • S2 cell inoculation Add culture medium, Y-27632 (Rocki), and then add PVA with a final concentration of 0.1 ⁇ 1mg/ml or a final concentration of 10-100ug/mL DS and 0.1 ⁇ 1mg/ml PVA in the culture device.
  • the suspension is inoculated into the culture device, and the culture device is shaken to evenly suspend the cells in the culture solution;
  • S3 cell medium exchange After culturing for 48 hours, change the medium with PVA-containing medium, and change the medium once a day at 60%;
  • step S1 When the hPSC is passaged, select a culture well with a coverage rate of 70%-80%, a regular edge of the clone and no differentiated cells, aspirate the medium and add 1 mL of calcium and magnesium-free PBS Wash the cells, add 1mL GCDR (cell dissociation reagent) after aspiration, put it back in the CO 2 incubator and incubate for 5-10 minutes, then aspirate the GCDR in the well, add 1mL mTeSR1 medium, pipette down the cells and pipette Into a single cell suspension.
  • GCDR cell dissociation reagent
  • the single cell suspension is a cell suspension of 500,000 single cells.
  • the medium in step S2 is a serum-free complete medium, preferably mTeSR1 medium.
  • the amount of Y-27632 (Rocki) added is 10uM.
  • the medium in step S3 is a serum-free complete medium, preferably mTeSR1 medium.
  • the final concentration of PVA in the PVA-containing medium is 0.1 to 1 mg/ml.
  • step S4 After the culture in step S4 is completed, centrifuge, discard the supernatant, add PBS to wash the cell aggregates, centrifuge, discard the supernatant, add trypsin, digest at room temperature for 9-12 minutes, add a serum-containing medium to terminate the digestion, and obtain cells.
  • the conditions of the centrifugation are centrifugation at 1500 rpm for 5 minutes; the trypsin is 0.25% trypsin.
  • the present invention combines low-concentration PVA with 40000kDA (D40) and 100ug/ml DS to use in vitro suspension culture of hPSCs.
  • the combined use of the two can improve the efficiency of cell expansion and keep the harvested cell aggregates small and uniform.
  • the size of the cell aggregates is relatively high, the center of the aggregates will not lack nutrients and oxygen due to the large size, and the recovered aggregates are easy to digest into single cells for passage or clinical use.
  • the present invention has the following advantages and beneficial effects:
  • the prior art uses DS to reduce cell aggregation by adjusting the charge on the cell surface to obtain high-quality small cell aggregates that are uniform in size and beneficial to recovery.
  • the cell expansion ratio of this technique has not been increased, that is, the number of cells still does not meet the requirements.
  • the present invention uses low-concentration PVA and 40000kDA (D40), 100ug/ml DS, which can not only reduce the size of cell aggregates and obtain uniform size aggregates, but also achieve a higher number of cells to meet the needs of in vitro expansion. The need for functional stem cells.
  • Figure 1 is a characterization diagram of cells obtained by 3D suspension culture + 100ug/mL DS + 1 mg/mL PVA in Example 1;
  • A is an image of aggregates after 5 days of culture, B is a comparison of the total number of cells after 5 days of culture, and C is 5
  • a comparison chart of the average diameter of the aggregates in the sky, D is the statistical comparison chart of the distribution interval of the aggregates in culture for 5 days;
  • Figure 2 is the characterization diagram of the cells obtained by 3D suspension culture + 100ug/mL DS + 0.1 mg/mL PVA in Example 2;
  • A is the image of aggregates after 5 days of culture, B is the comparison of the total number of cells after 5 days of culture, and C is the culture A comparison chart of the average diameter of aggregates in 5 days, D is a statistical comparison chart of the distribution interval of aggregates in culture for 5 days;
  • Figure 3 is a characterization diagram of the cells obtained by 3D suspension culture + 10ug/mL DS + 1 mg/mL PVA in Example 3;
  • A is an image of aggregates after 5 days of culture, B is a comparison of the total number of cells after 5 days of culture, and C is 5
  • a comparison chart of the average diameter of the aggregates in the sky, D is the statistical comparison chart of the distribution interval of the aggregates in culture for 5 days;
  • Figure 4 is a characterization diagram of cells obtained by 3D suspension culture of hPSC + dextran sulfate in Comparative Example 1.
  • the control group did not add DS, the experimental group 1 added 10ug/mL DS, and the experimental group 2 added 100ug/mL DS;
  • A was culture 5.
  • Day aggregate image B is the comparison chart of the total number of cells in culture for 5 days, C is the comparison chart of the average diameter of the aggregates in the culture for 5 days, D is the statistical comparison chart of the distribution of the aggregates in the culture for 5 days; E is the dryness of the qPCR test for 5 days of culture Comparison chart of gene expression results;
  • Figure 5 is a characterization diagram of cells obtained by 3D suspension culture of hPSC + polyvinyl alcohol (PVA) in Comparative Example 2.
  • the control group did not add PVA and the experimental group added 1 mg/mLPVA;
  • A is the image of the aggregates cultured for 5 days, and B is the cultured 5 Comparison of the total number of cells in a day, C is a comparison of the average diameter of aggregates in 5 days of culture, D is a statistical comparison of the distribution of aggregates in 5 days of culture;
  • E is a comparison of the results of qPCR detection of dry gene expression results in 5 days of culture.
  • Dextran Sulfate DS Manufacturer: SIGMA, Mr (relative molecular mass) ⁇ 10000, Concentration: 100ug/ml Storage Concentration: 100mg/ml That is, 500mgDS is fully dissolved in 5ml sterile water and stored at 4 degrees.
  • DS is added to the culture medium when the cells are inoculated, and 60% of the culture medium is replaced when the cells are cultured to 48 hours. The replaced culture medium does not need to be added with the used concentration of DS, that is, DS only acts for 48 hours.
  • PVA Manufacturer: ALDRICH, Mw 31000-500000, degree of hydrolysis 87%-89%.
  • Use concentration 0.1mg/ml, 0.5mg/ml, 1mg/ml.
  • Storage concentration 100mg/ml, that is, 1gPVA is fully dissolved in 1ml sterile water and stored at room temperature.
  • Low-concentration PVA 0.1-1mg/ml is added to the culture medium when cells are inoculated, and 60% of the culture medium is replaced when the cells are cultured for 48 hours. The replaced culture medium is continuously added with the concentration of PVA, that is, PVA acts on the entire culture cycle.
  • mice six-well plate; mTeSR1; GCDR; PBS; trypan blue; Y-27632; 0.25% pancreatin; Pluronic-F68; PVA; DS.
  • Example 1 3D suspension culture+100ug/mLDS+1mg/mL PVA
  • a method for improving the efficiency of in vitro suspension culture of human pluripotent stem cells which specifically includes the following steps:
  • Single-cell passage hPSC select a well of hPSC in good condition (good condition: 4-5 days after the passage of hPSC, the cell coverage will reach about 70%-80%, and the hPSC with regular edges and no differentiated cells will be cloned), and aspirate
  • good condition 4-5 days after the passage of hPSC, the cell coverage will reach about 70%-80%, and the hPSC with regular edges and no differentiated cells will be cloned
  • aspirate After discarding the medium, add 1mL of calcium and magnesium-free PBS to wash the cells, aspirate and add 1mL of GCDR, put it back in the CO 2 incubator and incubate for 5-10 minutes, then aspirate the GCDR in the well, add 1mL of mTeSR1 medium, pipette down the cells and use Pipette into a single cell suspension;
  • Figure 1 is the characterization diagram of the cells obtained by 3D suspension culture + 100ug/mLDS+1 mg/mL PVA in Example 1.
  • A is the image of aggregates after 5 days of culture
  • B is the comparison of the total number of cells after 5 days of culture
  • C is 5 days of culture.
  • D is a statistical comparison chart of the distribution interval of the aggregates in the 5-day culture.
  • Example 2 3D suspension culture+100ug/mLDS+0.1mg/mL PVA
  • a method for improving the efficiency of in vitro suspension culture of human pluripotent stem cells which specifically includes the following steps:
  • Single cell passage hPSC select a well of hPSC in good condition, aspirate the medium and add 1mL calcium-magnesium-free PBS to wash the cells, aspirate and add 1mL GCDR, put it back in the CO2 incubator and incubate for 5-10 minutes, then aspirate Discard the GCDR in the well, add 1 mL of mTeSR1 medium, blow down the cells and pipette into a single cell suspension;
  • Figure 2 is a characterization diagram of cells obtained by 3D suspension culture + 100ug/mLDS + 0.1 mg/mL PVA in Example 2; A is an image of aggregates cultured for 5 days, B is a comparison diagram of the total number of cells cultured for 5 days, and C is culture A comparison chart of the average diameter of aggregates in 5 days, and D is a statistical comparison chart of the distribution interval of aggregates in culture for 5 days.
  • Example 3 3D suspension culture + 10ug/mL DS + 1 mg/mL PVA
  • a method for improving the efficiency of in vitro suspension culture of human pluripotent stem cells which specifically includes the following steps:
  • Single cell passage hPSC select a well of hPSC in good condition, aspirate the medium and add 1mL calcium-magnesium-free PBS to wash the cells, aspirate and add 1mL GCDR, put it back in the CO2 incubator and incubate for 5-10 minutes, then aspirate Discard the GCDR in the well, add 1 mL of mTeSR1 medium, blow down the cells and pipette into a single cell suspension;
  • Figure 3 is a characterization diagram of cells obtained in Example 3, 3D suspension culture + 10ug/mLDS+1 mg/mLPVA; A is an image of aggregates after 5 days of culture, B is a comparison of the total number of cells after 5 days of culture, and C is 5 days of culture The average diameter of the aggregates is compared, and D is the statistical comparison of the distribution interval of the aggregates after 5 days of cultivation.
  • Comparative Example 1 3D suspension culture of hPSC + dextran sulfate (DS)
  • Single cell passage hPSC select a well of hPSC in good condition, aspirate the medium and add 1mL calcium-magnesium-free PBS to wash the cells, aspirate and add 1mL GCDR, put it back in the CO2 incubator and incubate for 5-10 minutes, then aspirate Discard the GCDR in the well, add 1 mL of mTeSR1 medium, blow down the cells and pipette into a single cell suspension;
  • Cell seeding aspirate 20ul of single cell suspension, stain with trypan blue and use a hemocytometer for counting; take out the well plate with Pluronic-F68, aspirate and discard the Pluronic-F68 dilution, add 5mL mTeSR1 and add 10uM Y-27632 (Rocki) to improve the survival rate of single cell seeding, in addition, add DS solution with final concentration of 10ug/mL and 100ug/mL to 2 wells respectively; draw the cell suspension containing 500,000 single cells and inoculate it into the wells In the medium, shake the well plate crisscross to make the cells evenly suspended in the culture medium;
  • Cell harvest After culturing to the 5th day, take photos of the aggregates and count the aggregate diameter; then collect the cell aggregates, centrifuge at 1500rpm for 5 minutes, discard the supernatant and add 1mL PBS to wash the cell aggregates, centrifuge at 1500rpm for 5 minutes, discard After the supernatant, add 1mL 0.25% pancreatin, digest at room temperature for 10 minutes and add 1mL serum-containing medium to stop the digestion.
  • Figure 4 is a characterization diagram of the cells obtained by 3D suspension culture of hPSC+dextran sulfate in Comparative Example 1.
  • the control group did not add DS, the experimental group 1 added 10ug/mL DS, and the experimental group 2 added 100ug/mL DS;
  • A was culture Image of aggregates in 5 days
  • B is the comparison of the total number of cells in the 5 days of culture
  • C is the comparison of the average diameter of the aggregates in the 5 days of culture
  • D is the statistical comparison of the distribution interval of the aggregates in the 5 days of culture
  • E is the 5 days of culture for qPCR detection dry Comparison chart of sex gene expression results.
  • Comparative Example 2 3D suspension culture of hPSC + polyvinyl alcohol (PVA)
  • Single cell passage hPSC select a well of hPSC in good condition, aspirate the medium and add 1mL calcium-magnesium-free PBS to wash the cells, aspirate and add 1mL GCDR, put it back in the CO2 incubator and incubate for 5-10 minutes, then aspirate Discard the GCDR in the well, add 1 mL of mTeSR1 medium, blow down the cells and pipette into a single cell suspension;
  • Cell seeding aspirate 20ul of single cell suspension, stain with trypan blue and use a hemocytometer for counting; take out the well plate with Pluronic-F68, aspirate and discard the Pluronic-F68 dilution, add 5mL mTeSR1 and add 10uM Y-27632 (Rocki) in order to improve the survival rate of single cell seeding, in addition, add PVA solution with a final concentration of 1mg/mL to the well; pipette the cell suspension containing 500,000 single cells and seed it into the well, cross and shake the well Plate so that the cells are evenly suspended in the culture medium;
  • Cell harvest On the 5th day of culture, take photos of the aggregates and count the aggregate diameter; then collect the cell aggregates, centrifuge at 1500rpm for 5 minutes, discard the supernatant and add 1mL PBS to wash the cell aggregates, centrifuge at 1500rpm for 5 minutes, discard After the supernatant, add 1mL 0.25% pancreatin, digest at room temperature for 10 minutes and add 1mL serum-containing medium to stop the digestion. After staining with trypan blue, use a hemocytometer to count; sample 3 times per well and count 3 times, and the final total number of cells is averaged Count; RNA is extracted from the remaining cells after counting, reverse transcribed into cDNA, and then qPCR is performed to detect the expression level of dryness genes.
  • Figure 5 is a characterization diagram of cells obtained by 3D suspension culture of hPSC + polyvinyl alcohol (PVA) in Comparative Example 2.
  • the control group did not add PVA and the experimental group added 1 mg/mLPVA;
  • A is the image of the aggregates cultured for 5 days, and B is the cultured 5 Comparison of the total number of cells in a day, C is a comparison of the average diameter of aggregates in 5 days of culture, D is a statistical comparison of the distribution of aggregates in 5 days of culture;
  • E is a comparison of the results of qPCR detection of dry gene expression results in 5 days of culture.

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Abstract

提供了一种人多功能干细胞体外悬浮培养的方法，具体为将人多功能干细胞用含聚乙烯醇或葡聚糖硫酸酯与聚乙烯醇的培养基进行三维培养；所述葡聚糖硫酸酯在培养基中的浓度为10～100ug/ml；所述聚乙烯醇在培养基中的浓度为0.1～1mg/ml。该方法能够减少细胞聚集体的大小且得到尺寸均一的聚集体，还能达到较高的细胞数量以满足体外扩增人多功能干细胞的需求，提高了人多功能干细胞的培养效率。

Description

一种提高人多功能干细胞体外悬浮培养的效率的方法 技术领域
本发明属于细胞培养的技术领域，具体涉及一种提高人多功能干细胞体外悬浮培养效率的方法。
背景技术
人胚胎干细胞(hESC)和人诱导的多功能干细胞(hiPSC)统称人多功能干细胞(hPSC))，在细胞治疗、组织工程和再生医学以及药物研发和生物技术应用上有着巨大的前景。传统的利用塑料培养板和异源培养基进行细胞扩增的二维(2D)培养技术扩增细胞效率有限，且长期传代扩增后，细胞会失去干性和分化能力。为了获得高数量和高质量的人多功能干细胞，应用了以生物反应器为基础的模拟生物体内三维环境(3D)培养技术进行人多功能干细胞的大规模扩增。目前，在生物反应器中以单细胞形式接种后以细胞聚集体形式进行扩增的策略与微载体培养策略相比，具有节约成本且细胞易于回收等优势，但该策略形成了尺寸较大且大小不均匀的团聚体。由于大聚集体(＞300μm)直径较大，营养物质及氧分难以扩散到聚集体中心而导致细胞分化或死亡。
聚集体大小控制是以聚集体方式培养细胞策略的一个关键参数，可以通过控制剪切力的物理方法以及加入小分子等化学方法控制聚集体尺寸以获得较均一的细胞聚集体产物。聚硫酸化合物广泛应用于生物制药行业，它们不仅能够通过调节细胞表面的电荷来减少细胞聚集，而且对细胞具有抗凋亡作用。同时有文献报道葡聚糖硫酸酯(DS)可以减少hPSCs的细胞聚集而不降低其多能性。Lipsitz YY等人[1Lipsitz YY,Tonge PD,Zandstra PW.Chemically controlled aggregation of pluripotent stem cells.Biotechnol Bioeng 2018；115(8):2061–6.]报道了40000kDA(D40)、100ug/mlDS条件下悬浮培养细胞能使得hPSCs聚集体保持较小、均一的尺寸进行扩增且多能性、核型保持稳定。Nogueira等人[Nogueira,D.E.S.,Rodrigues,C.A.V.,Carvalho,M.S.et al.Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel TM bioreactors.J Biol Eng 2019；13,74.]报道了利用一次性立式滚轮反应器结合100ug/mlDS获得(2.3±0.2)×10 6cells/mL的细胞且收获平均直径346±11μm的细胞聚集体，其多能性、核型保持稳定。虽然使用DS能通过调节细胞表面的电荷来减少细胞聚集，有利于得到尺寸均一、利于回收的高质量小细胞聚集体，但细胞扩增倍数未能得到提升即细胞数量仍然达不到要求。
本发明将DS(葡聚糖硫酸酯)与PVA组合使用，提高了干细胞体外悬浮培养的效率，在以细胞聚集体形式扩增策略中控制了细胞聚集体尺寸的大小并且提高了细胞的扩增倍数，所回收的细胞多能性能够得到维持，满足体外扩增的需求。PVA和DS的结合使用不仅能够得到尺寸均一的聚集体，还能达到较高的细胞数量以满足临床需求。
发明内容
针对以上技术的不足之处，本发明的目的是提供一种提高人多功能干细胞体外悬浮培养效率的方法。本发明使用PVA能够对hPSCs具有促增殖的作用，进一步地将DS(葡聚糖硫酸酯)与PVA组合使用，不仅能够得到尺寸均一的聚集体，还能达到较高的细胞数量，提高了干细胞体外悬浮培养的效率。本发明的方法能够大规模体外悬浮培养hPSC。
本发明的目的通过以下技术方案实现：
一种提高人多功能干细胞体外悬浮培养效率的方法，包括以下步骤：将人多功能干细胞用含PVA(聚乙烯醇)或DS(葡聚糖硫酸酯)与PVA(聚乙烯醇)的培养基进行三维培养。
所述DS(葡聚糖硫酸酯)在培养基中的浓度为10～100ug/ml，其分子量为40000kDA(D40)；
所述PVA在培养基中的浓度为0.1～1mg/ml。
当选用含DS(葡聚糖硫酸酯)与PVA(聚乙烯醇)的培养基进行三维培养时，DS(葡聚糖硫酸酯)在培养基中的浓度优选为100ug/ml，PVA在培养基中的浓度优选为1mg/ml。
所述培养基为无血清完全培养基，优选为mTeSR1培养基。
所述培养基中还添加了Y-27632(Rocki)。
三维培养48h后用含PVA的培养基进行换液，每天60％量换液一次。
所述提高人多功能干细胞体外悬浮培养效率的方法，具体包括以下步骤：
S1单细胞传代人多功能干细胞(hPSC)：在hPSC传代后，选取覆盖率达70％-80％，克隆边缘规整且无分化的细胞，并将其制成单细胞悬浮液；
S2细胞接种：在培养装置中依次加入培养基、Y-27632(Rocki)，随后加入终浓度为0.1～1mg/ml PVA或终浓度10-100ug/mLDS和0.1～1mg/ml PVA，将单细胞悬浮液接种至培养装置中，摇晃培养装置使细胞均匀悬浮在培养液中；
S3细胞换液：培养48h后，用含PVA的培养基进行换液，每天60％量换液一次；
S4收获细胞：培养5～6天，收集细胞。
步骤S2中所述培养装置为低粘附培养装置，如：商业化的低粘附孔板，采用常规方法制备的低粘附孔板：所述常规方法制备的粘附孔板具体步骤：采用DMEM/F12培养基稀释Pluronic-F68，获得稀释的Pluronic-F68(DMEM/F12:Pluronic-F68＝1:1(体积比))；将稀释的Pluronic-F68铺在孔板中，将孔板置于CO 2培养箱中孵育。
步骤S1中所述单细胞悬浮液具体制备步骤：在hPSC传代时，选取覆盖率达70％-80％，克隆边缘规整且无分化细胞的培养孔，吸弃培养基后加入1mL无钙镁PBS清洗细胞，吸弃后加入1mL GCDR(细胞解离试剂)，放回CO 2培养箱中孵育5-10分钟，之后吸弃孔中的GCDR，加入1mLmTeSR1培养基，将细胞吹打下来并用移液器吹成单细胞悬液。
步骤S2中单细胞悬浮液为50万单细胞的细胞悬液。步骤S2中所述培养基为无血清完全培养基，优选为mTeSR1培养基。
Y-27632(Rocki)的加入量为10uM。
步骤S3中所述培养基为无血清完全培养基，优选为mTeSR1培养基。
含PVA的培养基中PVA的终浓度为0.1～1mg/ml。
步骤S4中培养完成后，离心，弃上清液，加入PBS清洗细胞聚集体，离心，弃上清液后加入胰蛋白酶，室温消化9～12分钟，加入含血清培养基终止消化，获得细胞。所述离心的条件为1500rpm离心5分钟；所述胰蛋白酶选用0.25％胰蛋白酶。
本发明将低浓度PVA与40000kDA(D40)、100ug/ml DS相结合使用体外悬 浮培养hPSCs，两者的结合使用能够提高细胞扩增的效率，使得所收获的细胞聚集体保持较小、均一的尺寸，这种细胞聚集体质量较高，聚集体中心不会因为尺寸较大而缺乏养分以及氧气，并且所回收的聚集体易于消化成单细胞进而进行传代或者临床使用。
相对于现有技术，本发明具有如下优点及有益效果：
现有技术使用DS，通过调节细胞表面的电荷来减少细胞聚集，得到尺寸均一、利于回收的高质量小细胞聚集体，但是该技术细胞扩增倍数未能得到提升即细胞数量仍然达不到要求。而本发明使用低浓度的PVA与40000kDA(D40)、100ug/ml DS，不仅能够减少细胞聚集体的大小且得到尺寸均一的聚集体，还能达到较高的细胞数量以满足体外扩增人多功能干细胞的需求。
附图说明
图1为实施例1中3D悬浮培养+100ug/mL DS+1mg/mL PVA所获得细胞的表征图；A为培养5天聚集体图像，B为培养5天细胞总数对比图，C为培养5天聚集体平均直径对比图，D为培养5天聚集体分布分布区间统计对比图；
图2为实施例2中3D悬浮培养+100ug/mL DS+0.1mg/mL PVA所获得细胞的表征图；A为培养5天聚集体图像，B为培养5天细胞总数对比图，C为培养5天聚集体平均直径对比图，D为培养5天聚集体分布分布区间统计对比图；
图3为实施例3中3D悬浮培养+10ug/mL DS+1mg/mL PVA所获得细胞的表征图；A为培养5天聚集体图像，B为培养5天细胞总数对比图，C为培养5天聚集体平均直径对比图，D为培养5天聚集体分布分布区间统计对比图；
图4为对比例1中3D悬浮培养hPSC+葡聚糖硫酸酯所获得细胞的表征图，对照组不加DS，实验组1加10ug/mL DS，实验组2加100ug/mLDS；A为培养5天聚集体图像，B为培养5天细胞总数对比图，C为培养5天聚集体平均直径对比图，D为培养5天聚集体分布分布区间统计对比图；E为培养5天qPCR检测干性基因表达结果对比图；
图5为对比例2中3D悬浮培养hPSC+聚乙烯醇(PVA)所获得细胞的表征图，对照组不加PVA，实验组加入1mg/mLPVA；A为培养5天聚集体图像，B为培养5天细胞总数对比图，C为培养5天聚集体平均直径对比图，D为培养5天聚集体分布分布区间统计对比图；E为培养5天qPCR检测干性基因表达结果 对比图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。葡聚糖硫酸酯DS：厂商：SIGMA，Mr(相对分子质量)～10000，使用浓度：100ug/ml储存浓度：100mg/ml即500mgDS充分溶于5ml无菌水中4度保存。DS在接种细胞时添加入培养基中，细胞培养至48h时更换60％培养基，更换的培养基无需再添加使用浓度的DS即DS仅作用48h。
PVA：厂商：ALDRICH，Mw 31000-500000,水解度87％-89％。
使用浓度：0.1mg/ml，0.5mg/ml，1mg/ml。储存浓度：100mg/ml即1gPVA充分溶于1ml无菌水中常温保存。低浓度PVA(0.1-1mg/ml)在接种细胞时添加入培养基中，细胞培养至48h时更换60％培养基，更换的培养基持续添加使用浓度的PVA即PVA作用于整个培养周期。
实验材料：六孔板；mTeSR1；GCDR；PBS；台盼蓝；Y-27632；0.25％胰酶；Pluronic-F68；PVA；DS。
实施例1：3D悬浮培养+100ug/mLDS+1mg/mL PVA
一种提高人多功能干细胞体外悬浮培养效率的方法，具体包括以下步骤：
S1.预铺Pluronic-F68制备低粘附孔：在六孔板中以2mL/孔的体积预铺稀释好的Pluronic-F68(DMEM/F12:Pluronic-F68＝1:1)，将孔板放回CO 2培养箱中孵育1小时以上；
S2.单细胞传代hPSC：选取状态良好的hPSC一孔(状态良好：在hPSC传代后4-5天时，细胞覆盖率达70％-80％左右，克隆边缘规整且无分化细胞的hPSC)，吸弃培养基后加入1mL无钙镁PBS清洗细胞，吸弃后加入1mL GCDR，放回CO 2培养箱中孵育5-10分钟，之后吸弃孔中的GCDR，加入1mLmTeSR1培养基，将细胞吹打下来并用移液器吹成单细胞悬液；
S3.细胞接种：吸取20ul单细胞悬液，台盼蓝染色后利用血球计数板进行计数；取出预铺好Pluronic-F68的孔板，吸弃Pluronic-F68稀释液，加入5mL mTeSR1并加入10uM的Y-27632(Rocki)以提高单细胞接种的存活率，另外在孔中分别加入终浓度100ug/mL的DS溶液+1mg/mLPVA溶液；吸取含有50 万单细胞的细胞悬液并接种到孔中，十字交叉摇晃孔板以使细胞均匀悬浮在培养液中；
S4.细胞换液：在48h后每孔每天换液60％即3mLmTeSR1培养基以及加入对应浓度的PVA溶液(1mg/mL)，DS溶液不再加入培养基中；
S5.细胞收获：培养到第5天时，对聚集体进行拍照，统计聚集体直径；之后收集细胞聚集体，1500rpm离心5分钟，弃上清后加入1mLPBS清洗细胞聚集体，1500rpm离心5分钟，弃上清后加入1mL 0.25％胰酶，室温消化10分钟后加入1mL含血清培养基终止消化，台盼蓝染色后利用血球计数板进行计数；每孔取样3次并计数三次，最终细胞总数取平均数。
实验结果见图1，图1中对照组不加入PVA+DS，实验组加入100ug/mL的DS溶液+1mg/mLPVA。图1为实施例1中，3D悬浮培养+100ug/mLDS+1mg/mL PVA所获得细胞的表征图；A为培养5天聚集体图像，B为培养5天细胞总数对比图，C为培养5天聚集体平均直径对比图，D为培养5天聚集体分布分布区间统计对比图。
实施例2：3D悬浮培养+100ug/mLDS+0.1mg/mL PVA
一种提高人多功能干细胞体外悬浮培养效率的方法，具体包括以下步骤：
S1.预铺Pluronic-F68制备低粘附孔：在六孔板中以2mL/孔的体积预铺稀释好的Pluronic-F68(DMEM/F12:Pluronic-F68＝1:1),将孔板放回CO2培养箱中孵育1小时以上；
S2.单细胞传代hPSC：选取状态良好的hPSC一孔，吸弃培养基后加入1mL无钙镁PBS清洗细胞，吸弃后加入1mL GCDR，放回CO2培养箱中孵育5-10分钟，之后吸弃孔中的GCDR，加入1mLmTeSR1培养基，将细胞吹打下来并用移液器吹成单细胞悬液；
S3.细胞接种：吸取20ul单细胞悬液，台盼蓝染色后利用血球计数板进行计数；取出预铺好Pluronic-F68的孔板，吸弃Pluronic-F68稀释液，加入5mL mTeSR1并加入10uM的Y-27632(Rocki)以提高单细胞接种的存活率，另外在孔中分别加入终浓度100ug/mL的DS溶液+0.1mg/mL PVA溶液；吸取含有50万单细胞的细胞悬液并接种到孔中，十字交叉摇晃孔板以使细胞均匀悬浮在培养液中；
S4.细胞换液：在48h时每孔每天换液60％即3mLmTeSR1培养基以及加入对应浓度的PVA溶液，DS溶液不再加入培养基中；
S5.细胞收获：培养到第5天时，对聚集体进行拍照，统计聚集体直径；之后收集细胞聚集体，1500rpm离心5分钟，弃上清后加入1mLPBS清洗细胞聚集体，1500rpm离心5分钟，弃上清后加入1mL 0.25％胰酶，室温消化10分钟后加入1mL含血清培养基终止消化，台盼蓝染色后利用血球计数板进行计数；每孔取样3次并计数三次，最终细胞总数取平均数。
实验结果见图2，对照组不加入PVA+DS，实验组加入100ug/mL的DS溶液+0.1mg/mLPVA。图2为实施例2中，3D悬浮培养+100ug/mLDS+0.1mg/mL PVA所获得细胞的表征图；A为培养5天聚集体图像，B为培养5天细胞总数对比图，C为培养5天聚集体平均直径对比图，D为培养5天聚集体分布分布区间统计对比图。
实施例3：3D悬浮培养+10ug/mL DS+1mg/mL PVA
一种提高人多功能干细胞体外悬浮培养效率的方法，具体包括以下步骤：
S1.预铺Pluronic-F68制备低粘附孔：在六孔板中以2mL/孔的体积预铺稀释好的Pluronic-F68(DMEM/F12:Pluronic-F68＝1:1),将孔板放回CO2培养箱中孵育1小时以上；
S2.单细胞传代hPSC：选取状态良好的hPSC一孔，吸弃培养基后加入1mL无钙镁PBS清洗细胞，吸弃后加入1mL GCDR，放回CO2培养箱中孵育5-10分钟，之后吸弃孔中的GCDR，加入1mLmTeSR1培养基，将细胞吹打下来并用移液器吹成单细胞悬液；
S3.细胞接种：吸取20ul单细胞悬液，台盼蓝染色后利用血球计数板进行计数；取出预铺好Pluronic-F68的孔板，吸弃Pluronic-F68稀释液，加入5mL mTeSR1并加入10uM的Y-27632(Rocki)以提高单细胞接种的存活率，另外在孔中分别加入终浓度10ug/mL的DS溶液+1mg/mLPVA溶液；吸取含有50万单细胞的细胞悬液并接种到孔中，十字交叉摇晃孔板以使细胞均匀悬浮在培养液中；
S4.细胞换液：在48h时每孔每天换液60％即3mLmTeSR1培养基以及加入对应浓度的PVA溶液，DS溶液不再加入培养基中；
S5.细胞收获：培养到第5天时，对聚集体进行拍照，统计聚集体直径；之后收集细胞聚集体，1500rpm离心5分钟，弃上清后加入1mLPBS清洗细胞聚集体，1500rpm离心5分钟，弃上清后加入1mL 0.25％胰酶，室温消化10分钟后加入1mL含血清培养基终止消化，台盼蓝染色后利用血球计数板进行计数；每孔取样3次并计数三次，最终细胞总数取平均数。
实验结果见图3，对照组不加入PVA+DS，实验组加入10ug/mL的DS溶液+1mg/mL PVA。图3为实施例3中，3D悬浮培养+10ug/mLDS+1mg/mLPVA所获得细胞的表征图；A为培养5天聚集体图像，B为培养5天细胞总数对比图，C为培养5天聚集体平均直径对比图，D为培养5天聚集体分布分布区间统计对比图。
对比例1：3D悬浮培养hPSC+葡聚糖硫酸酯(DS)
实验材料：六孔板；mTeSR1；GCDR；PBS；台盼蓝；Y-27632；0.25％胰酶；Pluronic-F68；DS；
实验流程：1.配置DS储存溶液：称取500mg DS至15mL离心管中，加入5mL灭菌三蒸水，37℃水浴溶解，0.22um滤头过滤除菌，存于4℃冰箱待用；
2.预铺Pluronic-F68制备低粘附孔：在六孔板中以2mL/孔的体积预铺稀释好的Pluronic-F68(DMEM/F12:Pluronic-F68＝1:1),将孔板放回CO 2培养箱中孵育1小时以上；
3.单细胞传代hPSC：选取状态良好的hPSC一孔，吸弃培养基后加入1mL无钙镁PBS清洗细胞，吸弃后加入1mL GCDR，放回CO2培养箱中孵育5-10分钟，之后吸弃孔中的GCDR，加入1mLmTeSR1培养基，将细胞吹打下来并用移液器吹成单细胞悬液；
4.细胞接种：吸取20ul单细胞悬液，台盼蓝染色后利用血球计数板进行计数；取出预铺好Pluronic-F68的孔板，吸弃Pluronic-F68稀释液，加入5mL mTeSR1并加入10uM的Y-27632(Rocki)以提高单细胞接种的存活率，另外在2孔中分别加入终浓度为10ug/mL以及100ug/mL的DS溶液；吸取含有50万单细胞的细胞悬液并接种到孔中，十字交叉摇晃孔板以使细胞均匀悬浮在培养液中；
5.细胞换液：48h时每孔每天换液60％即3mL mTeSR1培养基以及加入对应浓度的DS溶液；
6.细胞收获：培养到第5天时，对聚集体进行拍照，统计聚集体直径；之后收集细胞聚集体，1500rpm离心5分钟，弃上清后加入1mLPBS清洗细胞聚集体，1500rpm离心5分钟，弃上清后加入1mL 0.25％胰酶，室温消化10分钟后加入1mL含血清培养基终止消化，台盼蓝染色后利用血球计数板进行计数；每孔取样3次并计数三次，最终细胞总数取平均数；计数后剩余细胞提取RNA，逆转录为cDNA后进行qPCR检测干性基因表达水平。
实验结果见图4。图4为对比例1中3D悬浮培养hPSC+葡聚糖硫酸酯所获得细胞的表征图，对照组不加DS，实验组1加10ug/mL DS，实验组2加100ug/mL DS；A为培养5天聚集体图像，B为培养5天细胞总数对比图，C为培养5天聚集体平均直径对比图，D为培养5天聚集体分布分布区间统计对比图；E为培养5天qPCR检测干性基因表达结果对比图。
对比例2：3D悬浮培养hPSC+聚乙烯醇(PVA)
实验材料：六孔板；mTeSR1；GCDR；PBS；台盼蓝；Y-27632；0.25％胰酶；Pluronic-F68；PVA；
实验流程：1.配置PVA储存溶液：称取1g PVA至15mL离心管中，加入10mL灭菌三蒸水，高压灭菌，常温储存待用；
2.预铺Pluronic-F68制备低粘附孔：在六孔板中以2mL/孔的体积预铺稀释好的Pluronic-F68(DMEM/F12:Pluronic-F68＝1:1),将孔板放回CO2培养箱中孵育1小时以上；
3.单细胞传代hPSC：选取状态良好的hPSC一孔，吸弃培养基后加入1mL无钙镁PBS清洗细胞，吸弃后加入1mL GCDR，放回CO2培养箱中孵育5-10分钟，之后吸弃孔中的GCDR，加入1mLmTeSR1培养基，将细胞吹打下来并用移液器吹成单细胞悬液；
4.细胞接种：吸取20ul单细胞悬液，台盼蓝染色后利用血球计数板进行计数；取出预铺好Pluronic-F68的孔板，吸弃Pluronic-F68稀释液，加入5mL mTeSR1并加入10uM的Y-27632(Rocki)以提高单细胞接种的存活率，另外 在孔中加入终浓度为1mg/mL的PVA溶液；吸取含有50万单细胞的细胞悬液并接种到孔中，十字交叉摇晃孔板以使细胞均匀悬浮在培养液中；
4.细胞换液：48h时每孔每天换液60％即3mL mTeSR1培养基以及加入对应浓度的PVA溶液；
5.细胞收获：培养到第5天时，对聚集体进行拍照，统计聚集体直径；之后收集细胞聚集体，1500rpm离心5分钟，弃上清后加入1mLPBS清洗细胞聚集体，1500rpm离心5分钟，弃上清后加入1mL 0.25％胰酶，室温消化10分钟后加入1mL含血清培养基终止消化，台盼蓝染色后利用血球计数板进行计数；每孔取样3次并计数三次，最终细胞总数取平均数；计数后剩余细胞提取RNA，逆转录为cDNA后进行qPCR检测干性基因表达水平。
实验结果见图5。图5为对比例2中3D悬浮培养hPSC+聚乙烯醇(PVA)所获得细胞的表征图，对照组不加PVA，实验组加入1mg/mLPVA；A为培养5天聚集体图像，B为培养5天细胞总数对比图，C为培养5天聚集体平均直径对比图，D为培养5天聚集体分布分布区间统计对比图；E为培养5天qPCR检测干性基因表达结果对比图。

Claims (10)

  1. 一种提高人多功能干细胞体外悬浮培养效率的方法，其特征在于：包括以下步骤：将人多功能干细胞用含聚乙烯醇或葡聚糖硫酸酯与聚乙烯醇的培养基进行三维培养。
  2. 根据权利要求1所述提高人多功能干细胞体外悬浮培养效率的方法，其特征在于：所述葡聚糖硫酸酯在培养基中的浓度为10～100ug/ml；
    所述聚乙烯醇在培养基中的浓度为0.1～1mg/ml。
  3. 根据权利要求1所述提高人多功能干细胞体外悬浮培养效率的方法，其特征在于：当选用含葡聚糖硫酸酯与聚乙烯醇的培养基进行三维培养时，葡聚糖硫酸酯在培养基中的浓度为100ug/ml，聚乙烯醇在培养基中的浓度为1mg/ml。
  4. 根据权利要求1所述提高人多功能干细胞体外悬浮培养效率的方法，其特征在于：葡聚糖硫酸酯的分子量为40000kDA；
    所述培养基为无血清完全培养基。
  5. 根据权利要求4所述提高人多功能干细胞体外悬浮培养效率的方法，其特征在于：所述培养基为mTeSR1培养基。
  6. 根据权利要求1所述提高人多功能干细胞体外悬浮培养效率的方法，其特征在于：所述培养基中还添加了Y-27632。
  7. 根据权利要求1所述提高人多功能干细胞体外悬浮培养效率的方法，其特征在于：三维培养48h后用含聚乙烯醇的培养基进行换液，每天60％量换液一次。
  8. 根据权利要求1～7任一项所述提高人多功能干细胞体外悬浮培养效率的方法，其特征在于：具体包括以下步骤：
    S1单细胞传代人多功能干细胞：在hPSC传代后，选取覆盖率达70％-80％，克隆边缘规整且无分化的细胞，并将其制成单细胞悬浮液；
    S2细胞接种：在培养装置中依次加入培养基、Y-27632，随后加入终浓度为0.1～1mg/ml PVA或终浓度10-100ug/mLDS和0.1～1mg/ml PVA，将单细胞悬浮液接种至培养装置中，摇晃培养装置使细胞均匀悬浮在培养液中；PVA：聚乙烯醇，DS：葡聚糖硫酸酯；
    S3细胞换液：培养48h后，用含PVA的培养基进行换液，每天60％量换液一次；
    S4收获细胞：培养5～6天，收集细胞。
  9. 根据权利要求8所述提高人多功能干细胞体外悬浮培养效率的方法，其特征在于：步骤S2中所述培养装置为低粘附培养装置；
    步骤S1中所述单细胞悬浮液具体制备步骤：在hPSC传代时，选取覆盖率达70％-80％，克隆边缘规整且无分化细胞的培养孔，吸弃培养基后加入1mL无钙镁PBS清洗细胞，吸弃后加入1mL GCDR，放回CO 2培养箱中孵育5-10分钟，之后吸弃孔中的GCDR，加入1mLmTeSR1培养基，将细胞吹打下来并用移液器吹成单细胞悬液；
    步骤S2中单细胞悬浮液为50万单细胞的细胞悬液；
    步骤S2中所述培养基为无血清完全培养基；
    步骤S3中所述培养基为无血清完全培养基；含PVA的培养基中PVA的终浓度为0.1～1mg/ml。
  10. 根据权利要求8所述提高人多功能干细胞体外悬浮培养效率的方法，其特征在于：步骤S4中培养完成后，离心，弃上清液，加入PBS清洗细胞聚集体，离心，弃上清液后加入胰蛋白酶，室温消化9～12分钟，加入含血清培养基终止消化，获得细胞。
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