WO2021098025A1 - 一种体外激活脂肪干细胞转化成原软骨细胞的方法 - Google Patents

一种体外激活脂肪干细胞转化成原软骨细胞的方法 Download PDF

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WO2021098025A1
WO2021098025A1 PCT/CN2019/130828 CN2019130828W WO2021098025A1 WO 2021098025 A1 WO2021098025 A1 WO 2021098025A1 CN 2019130828 W CN2019130828 W CN 2019130828W WO 2021098025 A1 WO2021098025 A1 WO 2021098025A1
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adipose
stem cells
medium
adipose stem
cell
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谢海涛
薛卫巍
王斌
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广东先康达生物科技有限公司
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Definitions

  • the invention relates to the field of medicine, in particular to a method for activating adipose stem cells to transform into protochondrocytes in vitro.
  • BMSCs bone marrow mesenchymal stem cells
  • MSCs mesenchymal stem cells
  • BMSCs bone marrow mesenchymal stem cells
  • Zuk et al. obtained multi-differentiated stem cells for the first time from adipose tissue suspension extracted by liposuction.
  • Adipose-derived stem cells (adipose-derived mesenchymalcells, ASCs) are a kind of stem cells superior to BMSCs. This is because stem cells extracted from adipose tissue have the advantages of easy extraction, large extraction volume, repeated extraction and rapid cell proliferation.
  • ASCs can differentiate into chondrocytes, osteoblasts, adipocytes, nerve cells, etc. in a specific microenvironment. Therefore, ASCs are expected to become good seed cells for tissue engineering.
  • the main problems are: (1) There are many kinds of known surface molecules of ASCs, but most of them cross with other mesenchymal stem cells, and there is no clear specific surface antigen. It is difficult to select the high specific surface marker antigen among them. Carry out combinatorial screening; (2) Although many laboratories try to use different materials and techniques to induce ASCs to differentiate into cartilage tissue, they cannot avoid the result of inflammation among cartilage tissues; (3) In many experimental chondrocytes, the later period The experimental results all showed the phenomenon of chondrocyte hypertrophy and proliferation; (4) At present, the induction of ASCs into chondrocytes is basically the construction of 3D models, but the clinical practicability of this method is not high.
  • the purpose of the present invention is to provide a method for activating the transformation of adipose stem cells into protochondrocytes in vitro.
  • a method for activating adipose stem cells to transform into protochondrocytes in vitro includes the following steps:
  • the DMEM medium ratio is 1% fetal bovine serum, 10ng/ml TGF- ⁇ , 100ng/ml BMP-6, 6.25 ⁇ g/ml insulin, 0.1 ⁇ mol/L dexamethasone, 6.25 ⁇ g/ml transfer Ferritin, 50 ⁇ mol/L ascorbyl phosphate;
  • the adipose-derived stem cells cultured for 7-9 days are injected into the joint cavity and transformed into protochondrocytes.
  • the steps of preparing the adipose-derived stem cells are:
  • adipose tissue Wash the adipose tissue with buffer solution to remove the remaining blood and tissue fragments, cut the cleaned adipose tissue into small pieces, digest them in a shaking box for 20-60 minutes, and then let it stand for stratification, and then suck the upper layer of fat cell fluid.
  • the adipose cell liquid is placed in a cabinet at 0-5 degrees Celsius and cultured for 1-2 hours, sealed and centrifuged, and the supernatant is removed to prepare adipose stem cell suspension;
  • adipose stem cell suspension Place the adipose stem cell suspension in the cell activation medium for activation and culture for 10-20 hours, so as to obtain the adipose stem cells that are initially expanded;
  • adipose stem cells Placing the adipose stem cells in at least one cell culture medium plus an activation reagent for culturing the adipose stem cell suspension for 12-15 hours, so as to obtain activated adipose stem cells;
  • adipose stem cells Take out the activated adipose stem cells from the culture medium, add physiological saline to clean them, and then add enzyme solution for digestion treatment. After the adipose stem cells grow to 80%-90% fusion, they will be cultured for three to six generations;
  • the subcultured culture solution is naturally placed for 3 to 5 hours to obtain micelles of adipose-derived stem cells.
  • adipose-derived stem cells cultured for 8 days are injected into the joint cavity to be transformed into protochondrocytes.
  • the adipose stem cells are cultured for five generations after they grow to 80%-90% fusion.
  • the cell activation medium is a serum-free lymphocyte medium supplemented with interleukin-2 and sapylin.
  • mammalian serum is added to the cell activation medium.
  • the mammalian serum is mammalian fetal serum.
  • the activation reagent is a calcium ionophore.
  • the calcium ionophore is selected from A23187 or ionomycin.
  • the present invention provides a method for activating adipose stem cells to transform into chondrocytes in vitro. Activate and stimulate it to the activated state to ensure the process of transforming to a cartilage cell, adjust the time point and intensity of activation, and then inject the activated ASCs into the diseased joint cavity to make it according to the original plan , Continue to transform into chondrocytes. This saves time, and no longer emphasizes the use of three-dimensional models and the difficult-to-control expansion growth environment, and the resulting cartilage cells also reduce the corresponding rejection.
  • the adipose tissue to be processed can be obtained from a patient or an immunologically acceptable donor, the adipose tissue is separated from the fat sample, and the activated adipose tissue is cultured .
  • An "immunologically acceptable donor" is a person with tissue, which includes adipose tissue.
  • the adipose tissue is washed with a buffer solution to remove residual blood and tissue fragments.
  • D-Hanks buffer solution is used to clean the adipose tissue.
  • the washed adipose tissue is cut into small pieces, digested in a shaking box for 20-60 minutes, and then statically cleaned. After it was stratified, the upper layer of fat cell fluid was sucked; and the fat cell fluid was placed in a 0-5 degrees Celsius box for 1-2 hours.
  • the cell activation medium can also be added with mammalian serum, wherein the mammalian serum is preferably mammalian fetal serum, such as fetal bovine serum, and the addition amount is about 10% and 15%.
  • the most basic nutrient for activating cells in vitro is a suitable medium.
  • These media generally consist of physiological saline, amino acids, vitamins and other compounds, which can be directly used by cells, preferably RPMI1640 medium or serum-free medium AIM-V.
  • the culture medium can be supplemented with mammalian immune serum, such as embryonic bovine immune serum.
  • Adipose stem cells are placed in at least one cell culture medium and an activation reagent is added to culture adipose stem cells for 12-15 hours to obtain activated adipose stem cells; in the method of the present invention Any cell culture medium can be used, but it is preferable to use the cell culture medium supplemented with calcium ionophore for 12-15 hours, wherein the calcium ionophore is selected from A23187 or ionomycin; in another embodiment, the activation reagent can also be selected Adenovirus.
  • the activated blood cells are washed with physiological saline at least once. In the embodiment of the present invention, it is preferably washed with physiological saline twice, and then digested with enzyme solution.
  • micellar immunogenic P5 generation ASCs cells After the adipose stem cells grow to 80% to 90%, they are passed through three to six times. Subculture, five generations are preferred in this example; the culture solution after subculture is naturally placed for 3 to 5 hours to obtain micellar immunogenic P5 generation ASCs cells, which are induced and activated in vitro under the action of various stimulating factors , Make it in the activated state.
  • the DMEM medium ratio is 1% fetal bovine serum, 10ng/ml TGF- ⁇ , 100ng/ml BMP-6, 6.25 ⁇ g/ml insulin, 0.1 ⁇ mol/L dexamethasone, 6.25 ⁇ g/ml transfer Ferritin, 50 ⁇ mol/L ascorbyl phosphate; adipose-derived stem cells forming micelles are placed in DMEM medium for culture; adipose-derived stem cells cultured for 7-9 days are injected into the joint cavity and transformed into protochondrocytes.
  • the relationship between the number of days of culture of ASCs and the activation state is that about 8 days of culture (7-9 days) ASCs is the best joint cavity injection period.
  • the ASCs maintain their original vitality and homing. It can accurately transform into the original chondrocytes. Within 6 days, ASCs are viable but the probability of transforming into original chondrocytes is small. If it is more than 9 days, the viability of ASCs will be weakened and the homing ability will decrease, resulting in a low conversion rate into the original chondrocytes. Therefore, the optimal activation time is determined to be around 8 days.
  • the inventor creatively invented the process of only activating in vitro while ensuring its original activity, stimulating it to the activated state, ensuring the process of transforming to a cartilage cell, adjusting the time point and intensity of activation, Then the activated ASCs are injected into the diseased joint cavity to continue to transform into chondrocytes according to the original plan. This saves time, and does not need to emphasize the use of three-dimensional models and the difficult-to-control expansion growth environment, and the produced cartilage cells also reduce the corresponding rejection reaction.
  • the cultivation temperature is, for example, between 30 and 42°C. In other embodiments, it is between 32 and 40°C, or between 37 and 38°C, or any range contained therein.
  • Those of ordinary skill in the art will recognize that other ranges of periods and temperatures within these explicit ranges are contemplated and are within the present disclosure. Or any range contained in it.
  • Those of ordinary skill in the art will recognize that other ranges of periods and temperatures within these explicit ranges are contemplated and are within the present disclosure. Or any range contained in it.
  • Those of ordinary skill in the art will recognize that other ranges of periods and temperatures within these explicit ranges are contemplated and are within the present disclosure.
  • the activated adipose stem cells can stimulate the secretion of exosomes after entering the human body, which is the secretion of other culture methods. 2-3 times.
  • these adipose stem cells can be activated.
  • the activated adipose stem cells have a functional level that can stimulate secretion, and the secretion of exosomes can be further increased by further subculture.
  • Calcium ionophore is a special metal ion substance that can freely pass through the lipid bilayer and soluble lipids.
  • ionophores There are two types of ionophores: ions formed by carriers or channels, like adenovirus as carriers, form a cage-like structure around special ions, which can diffuse freely in the hydrophobic region of the hydrophobic bilayer; ions formed by channels , Such as gram-positive bacteria, form a continuous liquid polar surface in the double-layer molecular membrane, allowing ions to diffuse through.
  • suitable ionophores of the present invention include calcium ionophore A23187 (calcomycin), sodium salt, magnesium salt and the like.
  • Calcium ionophore A23187 Such as calcium ionophore A23187, these carriers can respond to changes in pH gradient to accumulate calcium ions.
  • Calcium ionophore A23187 has an acidic carboxyl group, which can exchange with other cations throughout the biomembrane. When the ion exchange is completed, it will return to the other end of the membrane.
  • the effective concentration of ionophore is 0.05-0.5ug/ml, and the effective concentration of ionophore is the effective concentration for adipose stem cell activation.

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Abstract

提供了一种体外激活脂肪干细胞转化成原软骨细胞的方法，包括配置DMEM培养基，将形成微团的脂肪源干细胞置于DMEM培养基中进行培养，将培养7-9天的脂肪源干细胞注射进入关节腔转化为原软骨细胞。

Description

[根据细则26改正14.04.2020]　一种体外激活脂肪干细胞转化成原软骨细胞的方法 技术领域
本发明涉及医药领域，尤其是涉及一种体外激活脂肪干细胞转化成原软骨细胞的方法。
背景技术
至今较多运用的间充质干细胞(mesenchymalstemcells，MSCs)中骨髓间质干细胞(bonemarrowderivedstemcells，BMSCs)曾经是研究的热点，并被广泛应用于科学研究中。2001年，Zuk等从吸脂术抽取的脂肪组织悬液中第1次分离获得了多向分化的干细胞。脂肪源干细胞(adipose－derivedmesenchymalcells，ASCs)是一种优于BMSCs的干细胞，这是由于从脂肪组织提取的干细胞具有取材容易、取材量大、能反复取材及细胞增殖快速等优点。同时，ASCs可以在特定的微环境分别分化成软骨细胞、成骨细胞、脂肪细胞、神经细胞等。因此ASCs有望成为良好的组织工程种子细胞。
主要存在的问题是:(1)目前已知的ASCs表面分子种类繁多，但多数与其他间充质干细胞存在交叉，没有明确的特异性表面抗原，很难通过选择其中的高特异性表面标志抗原进行组合筛选；(2)虽然很多实验室尝试使用不同材料和技术诱导ASCs分化成软骨组织，但都避免不了软骨组织间出现炎症反应的结果；(3)在很多实验培养的软骨细胞，其后期实验结果都出现了软骨细胞肥大增生的现象；(4)目前诱导ASCs分化成软骨细胞基本上都是采取构建3D模型，但是这一方法的临床实用性不高。
发明内容
有鉴于此，针对目前干细胞转化为软骨细胞存在的问题，本发明目的在 于提供一种体外激活脂肪干细胞转化成原软骨细胞的方法。
本发明的目的是采用如下技术方案实现：
一种体外激活脂肪干细胞转化成原软骨细胞的方法，包括如下步骤：
配置DMEM培养基，所述DMEM培养基配比为1％胎牛血清、10ng/mlTGF－β、100ng/mlBMP－6、6.25μg/ml胰岛素、0.1μmol/L地塞米松、6.25μg/ml转铁蛋白、50μmol/L抗坏血酸磷酸酯；
将形成微团的脂肪源干细胞置于DMEM培养基中进行培养；
将培养7-9天的脂肪源干细胞注射进入关节腔转化为原软骨细胞。
可选的，所述脂肪源干细胞的制备步骤为：
利用缓冲液清洗脂肪组织去除残留的血液和组织碎片，将清洗后的脂肪组织剪成小块，在震荡箱中消化20min～60min，然后静置待其分层后，吸取上层脂肪细胞液，将脂肪细胞液置于0-5摄氏度的箱体内培养1-2小时，密封离心分离，去除上清液，制成脂肪干细胞悬液；
将脂肪干细胞悬液置于细胞激活培养基中进行激活培养10-20小时，以便得到初步扩增的脂肪干细胞；
将脂肪干细胞置于至少一种细胞培养基中加激活试剂进行脂肪干细胞悬液培养12-15小时，以便得到激活的脂肪干细胞；
将激活的脂肪干细胞从培养基中取出，加入生理盐水清洗干净，然后加入酶液消化处理，在脂肪干细胞长到80％～90％融合后传三到六代培养；
将传代后的培养液自然放置3～5小时得到微团的脂肪源干细胞。
可选的，将培养8天的脂肪源干细胞注射进入关节腔转化为原软骨细胞。
可选的，在脂肪干细胞长到80％～90％融合后传五代培养。
可选的，所述细胞激活培养基为添加了白介素-2和沙培林的无血清淋巴细胞培养基。
可选的，所述细胞激活培养基中添加了哺乳动物血清。
可选的，所述哺乳动物血清为哺乳动物胎血清。
可选的，所述激活试剂为钙离子载体。
可选的，所述钙离子载体选自A23187或离子霉素。
本发明提供的体外激活脂肪干细胞转化成原软骨细胞的方法为了充分利用有限的时间，尽快激活ASCs向软骨细胞转化，本发明人创造性地发明了在保证其原有的活性的同时，只在体外进行激活，将其刺激到激活状态，保证能向一个软骨细胞方向转变的过程，调整好激活的时间点和强度，再把激活状态的ASCs注射到病变的关节腔内，使其按照原定规划，继续向软骨细胞转化。这样做节约时间，不再强调使用三维模型和较难控制的扩增生长环境，产生的软骨细胞也减少相应的排斥反应。
具体实施方式
应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
一种体外激活脂肪干细胞转化成原软骨细胞的方法，根据本发明待处理的脂肪组织可以从患者或免疫学上可接受的供体获得，从脂肪样品中分离脂肪组织，并培养激活的脂肪组织。“免疫学上可接受的供体”是具有组织的人，其包括脂肪组织。首先利用缓冲液清洗脂肪组织去除残留的血液和组织碎片，在本实施例中使用D-Hanks缓冲液清洗，将清洗后的脂肪组织剪成小块，在震荡箱中消化20min～60min，然后静置待其分层后，吸取上层脂肪细胞液；并将脂肪细胞液置于0-5摄氏度的箱体内培养1-2小时，发明人发现利用前期在0-5摄氏度的箱体内培养1-2小时能增加脂肪细胞液的活性，再密封离心分离，去除上清液，制成脂肪干细胞悬液；将脂肪干细胞悬液置于细胞 激活培养基中进行激活培养10-20小时，以便得到初步扩增的脂肪干细胞；尽管可以在本发明的方法中可使用任何细胞激活培养基，但优选使用添加了白介素-2和沙培林的无血清淋巴细胞培养基培养10-20小时，以便得到初步扩增的脂肪干细胞；在另一实施例中细胞激活培养基还可以添加哺乳动物血清，其中的哺乳动物血清优选为哺乳动物胎血清，例如胎牛血清，添加量为10％和15％左右。体外激活细胞最基本的营养物质是合适的培养基。这些培养基一般由生理盐水、氨基酸、维生素和其他化合物组成，它们可以直接地被细胞利用，优选RPMI1640培养基或无血清培养基AIM-V。培养基可以补充哺乳动物免疫血清，如胚胎牛免疫血清将脂肪干细胞置于至少一种细胞培养基中加入激活试剂进行脂肪干细胞培养12-15小时，以便得到激活的脂肪干细胞；在本发明的方法中可使用任何细胞培养基，但优选使用添加了钙离子载体的细胞培养基培养12-15小时，其中的钙离子载体选自A23187或离子霉素；在另一实施例中激活试剂也可以选择腺病毒。将激活的血细胞经过至少一次的生理盐水清洗，在本发明实施例中优选经过二次的生理盐水清洗，然后加入酶液消化处理，在脂肪干细胞长到80％～90％融合后传三到六代培养，在本实施例中优选五代；将传代后的培养液自然放置3～5小时得到微团的免疫原性较低的P5代ASCs细胞，在各种刺激因子作用下在体外进行诱导激活，使其处在被激活的状态。配置DMEM培养基，所述DMEM培养基配比为1％胎牛血清、10ng/mlTGF－β、100ng/mlBMP－6、6.25μg/ml胰岛素、0.1μmol/L地塞米松、6.25μg/ml转铁蛋白、50μmol/L抗坏血酸磷酸酯；将形成微团的脂肪源干细胞置于DMEM培养基中进行培养；将培养7-9天的脂肪源干细胞注射进入关节腔转化为原软骨细胞。经实验证明，ASCs培养天数与激活状态之间的关系，在培养8天左右(7-9天)ASCs是最佳的关节腔注射期，此时ASCs既保持原有的活力和归巢性，又能准确的向原软骨细胞进行转化。在6天之间，ASCs活力有但是转化为原软骨细胞的几率小。如果多于9天，ASCs活力减弱归巢性降低，导致转化为原软骨细胞的转化率低。因此最佳的激活时间确定在8天左右。
本发明人创造性地发明了在保证其原有的活性的同时，只在体外进行激活，将其刺激到激活状态，保证能向一个软骨细胞方向转变的过程，调整好 激活的时间点和强度，再把激活状态的ASCs注射到病变的关节腔内，使其按照原定规划，继续向软骨细胞转化。这样做节约时间，不用强调使用三维模型和较难控制的扩增生长环境，产生的软骨细胞也减少相应的排斥反应。
培养温度例如在30和42℃之间。在其他实施方案中，在32和40℃之间，或在37和38℃之间，或其中包含的任何范围。本领域普通技术人员将认识到，在这些明确范围内的其他范围的时期和温度是预期的，并且在本公开内。或其中包含的任何范围。本领域普通技术人员将认识到，在这些明确范围内的其他范围的时期和温度是预期的，并且在本公开内。或其中包含的任何范围。本领域普通技术人员将认识到，在这些明确范围内的其他范围的时期和温度是预期的，并且在本公开内。
本发明涉及的含有脂肪干细胞的制备中本发明人发现当在其中加入钙离子载体或腺病毒后，被激活的脂肪干细胞进入人体后可以激发外泌体的分泌量，是其它培养方式分泌量的2-3倍。通过使脂肪干细胞与有激活试剂接触，可以激活这些脂肪干细胞，被激活的脂肪干细胞具有可以激发分泌的功能水平，并通过进一步传代培养使其外泌体的分泌量进一步增多。
钙离子载体是一种特殊金属离子物质，它可以自由通过脂质双分子层和可溶性脂质。有两种离子载体：载体或通道形成的离子，像腺病毒为载体，在特殊的离子周围形成一个像笼状般的结构，可以在疏水性双分子层疏水区进行自由扩散；通道形成的离子，如革兰氏阳性菌，在双层分子膜中形成连续的液体的极面，允许离子扩散通过。另外，除了上述所述的载体外，本发明适合的离子载体包括钙离子载体A23187(钙霉素)、钠盐、镁盐等。如钙离子载体A23187，这些载体能够对PH梯度的变化作出反应，以聚集钙离子。钙离子载体A23187具有一个酸性羧基团，它可以在整个生物膜中与其他阳离子进行交换，当离子交换完成之后，则回到膜的另一端。离子载体的有效浓度在0.05-0.5ug/毫升，离子载体的有效浓度是脂肪干细胞激活的有效浓度。
上面对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护 的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。

Claims (9)

  1. 一种体外激活脂肪干细胞转化成原软骨细胞的方法，其特征在于包括如下步骤：
    配置DMEM培养基，所述DMEM培养基配比为1％胎牛血清、10ng/ml TGF－β、100ng/ml BMP－6、6.25μg/ml胰岛素、0.1μmol/L地塞米松、6.25μg/ml转铁蛋白、50μmol/L抗坏血酸磷酸酯；
    将形成微团的脂肪源干细胞置于DMEM培养基中进行培养；
    将培养7-9天的脂肪源干细胞注射进入关节腔转化为原软骨细胞。
  2. 根据权利要求1所述的方法，其特征在于：所述脂肪源干细胞的制备步骤为：
    利用缓冲液清洗脂肪组织去除残留的血液和组织碎片，将清洗后的脂肪组织剪成小块，在震荡箱中消化20min～60min，然后静置待其分层后，吸取上层脂肪细胞液，将脂肪细胞液置于0-5摄氏度的箱体内培养1-2小时，密封离心分离，去除上清液，制成脂肪干细胞悬液；
    将脂肪干细胞悬液置于细胞激活培养基中进行激活培养10-20小时，以便得到初步扩增的脂肪干细胞；
    将脂肪干细胞置于至少一种细胞培养基中加激活试剂进行脂肪干细胞悬液培养12-15小时，以便得到激活的脂肪干细胞；
    将激活的脂肪干细胞从培养基中取出，加入生理盐水清洗干净，然后加入酶液消化处理，在脂肪干细胞长到80％～90％融合后传三到六代培养；
    将传代后的培养液自然放置3～5小时得到微团的脂肪源干细胞。
  3. 根据权利要求1所述的方法，其特征在于：将培养8天的脂肪源干细胞注射进入关节腔转化为原软骨细胞。
  4. 根据权利要求2所述的方法，其特征在于：在脂肪干细胞长到80％～90％融合后传五代培养。
  5. 根据权利要求2所述的方法，其特征在于：所述细胞激活培养基为添加了白介素-2和沙培林的无血清淋巴细胞培养基。
  6. 根据权利要求2所述的方法，其特征在于：所述细胞激活培养基中添加了哺乳动物血清。
  7. 根据权利要求7所述的方法，其特征在于：所述哺乳动物血清为哺乳动物胎血清。
  8. 根据权利要求2所述的方法，其特征在于：所述激活试剂为钙离子载体。
  9. 根据权利要求9所述的方法，其特征在于：所述钙离子载体选自A23187或离子霉素。
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