WO2021190620A1 - 细胞保存液及其试剂盒 - Google Patents

细胞保存液及其试剂盒 Download PDF

Info

Publication number
WO2021190620A1
WO2021190620A1 PCT/CN2021/083169 CN2021083169W WO2021190620A1 WO 2021190620 A1 WO2021190620 A1 WO 2021190620A1 CN 2021083169 W CN2021083169 W CN 2021083169W WO 2021190620 A1 WO2021190620 A1 WO 2021190620A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
preservation solution
cell preservation
stem cells
injection
Prior art date
Application number
PCT/CN2021/083169
Other languages
English (en)
French (fr)
Inventor
胡赓熙
Original Assignee
上海我武干细胞科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海我武干细胞科技有限公司 filed Critical 上海我武干细胞科技有限公司
Priority to CN202180023404.0A priority Critical patent/CN115315182A/zh
Publication of WO2021190620A1 publication Critical patent/WO2021190620A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a cell preservation solution and a kit thereof.
  • Stem cells are a type of multipotent cells with self-replication ability, and under certain conditions, they can differentiate into a variety of functional cells. Embryonic stem cells are totipotent stem cells with the strongest differentiation ability. However, due to their own tumorigenicity, ethical issues and other factors, their clinical application prospects are currently uncertain.
  • Mesenchymal stem cells are members of the stem cell family. They exist in a variety of tissues (such as bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue, adipose tissue, etc.), and have multidirectional differentiation potential.
  • Such stem cells have the potential to differentiate into a variety of mesenchymal cells (such as osteoblasts, cartilage and adipocytes, etc.) or non-mesenchymal cells, and have unique cytokine secretion functions, as well as immune regulation and anti-inflammatory
  • mesenchymal cells such as osteoblasts, cartilage and adipocytes, etc.
  • non-mesenchymal cells such as osteoblasts, cartilage and adipocytes, etc.
  • the effective cells have been reported to be used in the treatment of immune-related diseases, such as systemic lupus erythematosus, Crohn’s disease, graft-versus-host disease; some basic research and clinical trials have proved that mesenchymal stem cells can alleviate atherosclerosis .
  • mesenchymal stem cells directly affects its therapeutic effect. How to realize the convenience of long-term storage and transportation on the basis of ensuring its biological activity has become a key issue for mesenchymal stem cell drugs.
  • Physiological saline or glucose solution as a mesenchymal stem cell preservation solution due to nutrient deficiency, tension or isotonicity of the solution, etc. causes the cells to experience low viability, large amounts of swelling and death, and increased cell agglomeration rates during long-term storage or transportation.
  • Preservation solutions containing fetal bovine serum or cell culture media will endocytose the fetal bovine serum when the cells are in contact with the fetal bovine serum, which will cause changes in the expression characteristics of certain proteins of the cells, and may cause allergic reactions after the cells are returned to the human body.
  • the newly developed preservation solution has improved the problems of low cell biological activity and the use of fetal bovine serum compared with the previous preservation solution, but it has the problems of complicated preparation process, short cell preservation time, and inconvenient transportation.
  • the mesenchymal stem cell preservation solution with stable ingredients, diversified nutrients, close to blood osmotic pressure, strong buffering capacity, and long storage time and easy to transport is one of the problems that need to be solved urgently in the research and development of mesenchymal stem cell drugs.
  • the present invention provides a cell preservation solution, which is characterized in that the cell preservation solution contains human serum albumin, glucose, vitamin C, sodium lactate Ringer injection and compound amino acid injection.
  • the cell preservation solution also contains human serum.
  • the present invention also provides a cell preparation, which comprises the cell preservation solution of the present invention and the cells stored therein.
  • the cells are stem cells, preferably mesenchymal stem cells, such as umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, uterine membrane mesenchymal stem cells, and hair follicle mesenchymal stem cells.
  • the present invention also provides a kit containing:
  • the cell diluent is selected from: compound sodium chloride injection or physiological saline.
  • the present invention also provides another kit, which contains:
  • the cell diluent is selected from: compound sodium chloride injection or physiological saline.
  • Figure 1 shows the results of the adipogenic and osteogenic differentiation capacity of the hair follicle mesenchymal stem cells HF015 in Example 2 stored at 4°C for 120 hours.
  • Figure 2 shows the results of the reattachment ability of the hair follicle mesenchymal stem cells HF015 in Example 2 stored at 4°C for 120 hours.
  • Fig. 3 shows the results of flow cytometric apoptosis detection of hair follicle mesenchymal stem cells HF015 stored at 4°C for 120 hours in Example 2.
  • the “range” disclosed herein takes the form of the lower limit and the upper limit. There can be one or more lower limits, and one or more upper limits, respectively.
  • the given range is limited by selecting a lower limit and an upper limit.
  • the selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this way are inclusive and combinable, that is, any lower limit can be combined with any upper limit to form a range.
  • the present invention first provides a cell preservation solution, which is characterized in that it contains a mixture of human serum albumin (HSA), glucose, vitamin C, sodium lactate Ringer injection and compound amino acid injection.
  • HSA human serum albumin
  • the cell preservation solution may also contain other components that are harmless to cells and humans, such as pharmaceutically acceptable carriers, such as diluents such as physiological saline, and pH regulators such as buffers.
  • pharmaceutically acceptable carriers such as diluents such as physiological saline
  • pH regulators such as buffers.
  • the cell preservation solution is composed of human serum albumin, glucose, vitamin C, sodium lactate Ringer injection and compound amino acid injection.
  • the cell preservation solution comprises human serum albumin, glucose, vitamin C, human serum, sodium lactate Ringer injection, and compound amino acid injection.
  • the cell preservation solution is composed of human serum albumin, glucose, vitamin C, human serum, sodium lactate Ringer injection and compound amino acid injection.
  • the human serum albumin (HSA), glucose, vitamin C, human serum, sodium lactate Ringer injection and compound amino acid injection used in the present invention can be purchased from commercial sources, or can be prepared by using any existing technology.
  • the mass volume percentage concentration of human serum albumin in the cell preservation solution is 1%-8%, preferably 3%-7%, most preferably 4%-7%.
  • the mass volume percentage concentration of glucose in the cell preservation solution is 0.05%-2.5%, preferably 0.05%-1%, most preferably 0.125%.
  • the mass volume percentage concentration of vitamin C in the cell preservation solution is 0.01%-0.2%, preferably 0.05%-0.2%, most preferably 0.1%.
  • the volume percentage of human serum in the cell preservation solution is 10%-60%, preferably 10%-30%, most preferably 30%.
  • the volume ratio of sodium lactate Ringer injection and compound amino acid injection in the cell preservation solution is 1:1-1:1.5, preferably 1:1, which can be used as a cell preservation background solution.
  • the mass volume percent concentration (m/v) mentioned in this article refers to the mass grams (g) of the specified component per hundred milliliters (100 mL) of the solution.
  • a glucose mass volume percentage (m/v) of 0.05%-2.5% means that the mass of glucose powder contained in 100 ml of cell preservation solution is 0.05g-2.5g.
  • the volume percentage (v/v) refers to the percentage of the volume of the specified component in the unit volume of the solution.
  • a human serum volume percentage (v/v) of 10%-60% means that 100 ml of cells are stored The volume of the liquid containing human serum is 10mL-60mL.
  • the pH of the cell preservation solution is 5.5-7.5, preferably the pH is 6.0-6.5.
  • the osmotic pressure of the cell preservation solution is 240-360 mOsmol/kg (millosmolar concentration); preferably 280-320 mOsmol/kg (millosmolar concentration).
  • water for injection can be used to adjust the ion concentration/strength of the cell preservation solution, thereby adjusting its osmotic pressure.
  • the sodium lactate Ringer injection used in the present invention meets the requirements of the Pharmacopoeia.
  • the sodium lactate Ringer injection contains sodium lactate, sodium chloride, potassium chloride, and calcium chloride.
  • the sodium lactate Ringer injection contains components per 1000 mL: sodium lactate 3.10 g, sodium chloride 6.00 g, potassium chloride 0.30 g, and calcium chloride 0.20 g.
  • calcium chloride is preferably CaCl 2 ⁇ 2H 2 O.
  • the compound amino acid injection used in the present invention meets the requirements of the pharmacopoeia.
  • the compound amino acid injection contains L-proline, L-serine, L-alanine, L-isoleucine, L-leucine, L-aspartic acid, L -Tyrosine, L-glutamic acid, L-phenylalanine, L-arginine hydrochloride, L-lysine hydrochloride, L-valine, L-threonine, L- Histidine hydrochloride, L-tryptophan, L-methionine, L-cystine, glycine, sorbitol, sodium bisulfite.
  • the compound amino acid injection contains components per 1000 mL: L-proline 1.00g, L-serine 1.00g, L-alanine 2.00g, L-isoleucine 3.52g, L -Leucine 4.90g, L-aspartic acid 2.50g, L-tyrosine 0.25g, L-glutamic acid 0.75g, L-phenylalanine 5.33g, L-arginine hydrochloride 5.00 g, L-lysine hydrochloride 4.30g, L-valine 3.60g, L-threonine 2.50g, L-histidine hydrochloride 2.50g, L-tryptophan 0.90g, L- Methionine 2.25g, L-cystine 0.10g, Glycine 7.60g, Sorbitol 50.00g, Sodium bisulfite 0.5g.
  • the cell preservation solution of the present invention can be used to preserve various types of cells, and is particularly suitable for preservation of stem cells, especially mesenchymal stem cells, such as umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, uterine membrane mesenchymal stem cells and hair follicle mesenchyme Cytoplasmic stem cells.
  • mesenchymal stem cells such as umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, uterine membrane mesenchymal stem cells and hair follicle mesenchyme Cytoplasmic stem cells.
  • the present invention also provides a cell preparation, which contains the cell preservation solution of the present invention and the cells stored therein.
  • the cells are stem cells, preferably mesenchymal stem cells, such as umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, uterine membrane mesenchymal stem cells, and hair follicle mesenchymal stem cells.
  • the cell density in the cell preparation is 5 ⁇ 10 5 -2 ⁇ 10 7 cells/mL, more preferably 2 ⁇ 10 6 -2 ⁇ 10 7 cells/mL, which can be adjusted according to clinical needs.
  • the storage temperature of the stem cell preparation is preferably below 4°C, preferably 0-4°C, and the storage time is preferably 1-7 days, such as 1-5 days.
  • the viability of mesenchymal stem cells can still be maintained above 80%, even better, up to 90% after being stored in the cell preservation solution provided by the present invention for 120 hours, or even 180 hours.
  • cell viability refers to the ratio of the number of living cells to the total number of cells.
  • the method for preparing the cell preservation solution includes the following steps:
  • the present invention also discloses a kit containing:
  • the cell diluent is selected from: compound sodium chloride injection or physiological saline.
  • the present invention also provides another kit, which contains
  • the cell diluent is selected from: compound sodium chloride injection or physiological saline.
  • the compound sodium chloride injection used as a cell diluent contains sodium chloride, potassium chloride, and calcium chloride.
  • the compound sodium chloride injection contains 8.50 g of sodium chloride, 0.30 g of potassium chloride, and 0.33 g of calcium chloride per 1000 mL.
  • Physiological saline is a sodium chloride solution commonly used in physiological experiments or clinical practice with an osmotic pressure substantially equal to that of animal or human plasma, such as 0.9% (w/v) sodium chloride solution.
  • the kit further includes instructions for use, teaching that the cell preparation of the present invention or the cell preparation prepared with the cell preservation solution of the present invention should be diluted according to a certain dilution ratio with the cell diluent.
  • the dilution ratio may be 1:40-1:5, preferably 1:20-1:10.
  • the instructions for use also teach that the diluted cell preservation solution should be placed at room temperature and re-warmed and then returned to the human body for a time of 1-24 hours, preferably 1-10 hours, more preferably 5 hours.
  • the preparation method of mesenchymal stem cells used in the examples is as follows:
  • the preparation method of umbilical cord mesenchymal stem cells refers to the literature: Han Hua, Xue Gai, Zhang Junqin, etc. "Isolation, culture and identification of human umbilical cord mesenchymal stem cells” [ J]. Journal of Hebei Medical University, 2015, 36(1): 21-23; Reference literature for preparation methods of uterine mesenchymal stem cells: Zhou Yunfan, Yang Bo, Hu Xiang, et al. "Menstrual blood-derived endometrial mesenchymal stem cells Isolation, culture and identification of human adipose-derived stem cells”[J].
  • Example 1 Detection of viability of mesenchymal stem cells preserved in cell preservation solutions of different formulas
  • the preparation method is to first add human serum albumin, glucose and vitamin C powder to step 1) background solution and mix well, then add human serum (AB type, purchased from Shanghai Hengyuan Biotechnology Co., Ltd.), use step 1)
  • human serum (AB type, purchased from Shanghai Hengyuan Biotechnology Co., Ltd.)
  • the prepared background solution is made to a constant volume of 1L, and after mixing it evenly, it is sucked with a 20mL syringe and filtered and sterilized with a 0.22 ⁇ m filter.
  • Four cell preservation solutions with different formulations were prepared (Table 1).
  • the prepared cell preparation is stored in a refrigerator at 4°C at low temperature. After storage for 180 hours, use AO/PI (AO (Acridine Orange), PI (Propidium Iodide) Propidium Iodide) double staining cell apoptosis detection kit (DNA probe double staining cell nucleus method, purchased from Shanghai Rui Yu Biological Technology Co., Ltd.) tested the cell viability of mesenchymal stem cells stored in cell preservation solutions of different formulations. The results are shown in Table 2.
  • AO/PI AO (Acridine Orange)
  • PI Propidium Iodide
  • Propidium Iodide Propidium Iodide
  • This ratio refers to the volume ratio of sodium lactate Ringer injection: compound amino acid injection in the background solution
  • the cell preservation solution of the present invention has good preservation for umbilical cord, fat, hair follicle and uterine mesenchymal stem cells. After 180 hours of storage, the cell viability of the four types of cells can still be maintained above 80%, and the viability of some cultured cells even exceeds 90%.
  • Example 2 Detection of cell performance after storing hair follicle mesenchymal stem cells in the cell preservation solution for 120 hours
  • the hair follicle-derived mesenchymal stem cells (numbered "HF015") stored in the cell preservation solution of formula 1 as described in Example 1 were taken for 0 hours, 96 hours, and 120 hours later to test the cell viability.
  • AO/PI double staining cell apoptosis detection kit was used for detection, and the results are shown in Table 3.
  • hair follicle-derived mesenchymal stem cells HF015 stored in the cell preservation solution of formula 1 as described in Example 1 for 0 hours, 96 hours, and 120 hours, with a cell concentration of 1.0 ⁇ 10 5 cells/well.
  • the hair follicle mesenchymal stem cells are evenly spread on the coated 12-well plate, placed in a 5% CO 2 , 37°C incubator, and cultured for 3-4 days until the cell fusion rate reaches 90%-100%.
  • osteogenic differentiation complete medium purchased from Saiye Biotechnology Co., Ltd.
  • change the medium every 3 days and use Alizarin Red S (purchased from Beijing Soleibao Technology Co., Ltd.) stained osteogenic differentiated cells.
  • Alizarin Red S purchased from Beijing Soleibao Technology Co., Ltd.
  • Mesenchymal stem cells are evenly spread on the coated twelve-well plate, and placed in a 5% CO 2 , 37°C incubator for 3-4 days until the cell fusion rate is about 90%-100%, and the wells are aspirated and discarded in an ultra-clean table Inner medium, add 1ml adipogenic differentiation medium (purchased from STEMCELL), change the medium every 3 days, and stain with oil red O when adipogenic differentiation is induced for 14 days (purchased from Beijing Solebold Technology Co., Ltd.) Stain adipogenic differentiated cells. The results are shown in Table 3 and Figure 1.
  • Table 3 shows the test results of each cell performance.
  • the hair follicle-derived mesenchymal stem cells in the cell preservation solution are stored at 4°C for 120 hours, the cell viability rate is greater than 90%, the flow cytometric apoptosis detection viability rate is greater than 90%, and the cell reattachment ability is greater than 20%; and 0 Compared with hours, there was no significant change in the adipogenic and osteogenic differentiation ability of cells after 96 hours and 120 hours of low temperature storage. Therefore, the hair follicle-derived mesenchymal stem cells can be stored stably for 120 hours in the cell storage solution at 4°C without affecting the cell viability and differentiation potential.
  • Example 3 Detection of the viability of mesenchymal stem cells stored in cell preservation solutions with different concentrations of glucose
  • the prepared cell preparation is stored in a refrigerator at 4°C at low temperature. After 180 hours of storage, the AO/PI double-stained cell apoptosis detection kit was used to detect the cell viability of mesenchymal stem cells preserved in cell preservation solutions with different concentrations of glucose. The results are shown in Table 5.
  • This ratio refers to the volume ratio of sodium lactate Ringer injection: compound amino acid injection in the background solution
  • Example 4 Detection of cell viability of cell preservation solutions diluted with different dilution preparations after being stored at room temperature for 5 hours
  • Mesenchymal stem cells were prepared in formula 1 cell preservation solution as described in Example 1. After being stored at 4°C for 24 hours or 48 hours, they were diluted with 1:10 (such as sodium lactate Ringer injection, compound amino acid injection). Solution, normal saline or 5% glucose injection), placed at room temperature for 5 hours, using AO/PI double staining cell apoptosis detection kit to detect cell viability. The results are shown in Table 6.
  • the compound amino acid injection is used as the diluent, the osmotic pressure of the cell preservation solution will be too high, which is not conducive to the preservation of cells. Therefore, in the present invention, physiological saline or compound sodium chloride injection is used as the diluent to prepare the cell preservation solution.
  • Example 5 Detection of the viability of mesenchymal stem cells stored in serum-free cell preservation solutions with different concentrations of human albumin
  • the preparation method is to add the powder of human serum albumin, glucose and vitamin C to step 1) the background solution and mix well, then add the human serum (if any), use the background solution and injection water prepared in step 1) to press The volume ratio is 2:3 and the resulting solution is mixed to a constant volume of 1L. After the mixture is evenly mixed, it is sucked with a 20mL syringe and filtered and sterilized with a 0.22 ⁇ m filter. Six cell preservation solutions with different formulations were prepared, and their pH and osmotic pressure were measured. (Table 7).
  • the centrifuged hair follicles and mesenchymal stem cells of the uterine membrane were resuspended in the above-prepared cell preservation solutions of different formulas, and the final cell density was 2 ⁇ 10 6 -5 ⁇ 10 6 cells/mL.
  • the prepared cell preparation is stored in a refrigerator at 4°C at low temperature. After 144 hours of storage, the Annexin V-FITC/PI Apoptosis Kit (Hangzhou Lianke Biotechnology Co., Ltd.) was used to detect the cell viability of hair follicle-derived mesenchymal stem cells stored in cell preservation solutions of different formulations. The results are shown in the table. 8.
  • the above-mentioned cell preservation solutions with different concentrations of human albumin have good preservation ability for both mesenchymal stem cells.
  • the cell viability of the two mesenchymal stem cells can basically be maintained above 90% after 144 hours of storage.
  • the serum-free cell preservation solution of human albumin with a concentration of 4%-7% by mass and volume can also maintain the viability of mesenchymal stem cells; and, the preservation of serum-free cells In the liquid, the mass volume percentage concentration of human albumin is preferably 5%.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

本发明公开了一种细胞保存液及其试剂盒。所述细胞保存液包含人血清白蛋白、葡萄糖、维生素C、乳酸钠林格注射液、复方氨基酸注射液和可选的人血清；优选地，所述细胞保存液由人血清白蛋白、葡萄糖、维生素C、乳酸钠林格注射液、复方氨基酸注射液和可选的人血清组成。本发明还公开了由所述保存液配制的细胞制剂及其试剂盒。本发明的细胞保存液尤其适用于干细胞等细胞样品的保存。

Description

细胞保存液及其试剂盒 技术领域
本发明属于生物技术领域，具体涉及一种细胞保存液及其试剂盒。
背景技术
干细胞是一类具有自我复制能力的多潜能细胞，在一定条件下，可以分化成多种功能细胞。胚胎干细胞是分化能力最强的全能干细胞，然而因其自身成瘤性、伦理问题等多种因素的影响，其临床应用前景目前并不明朗。间充质干细胞(mesenchymal stem cells,MSCs)是干细胞家族的成员之一，存在于多种组织(如骨髓、脐带血和脐带组织、胎盘组织、脂肪组织等)，具有多向分化潜力。这类干细胞具有向多种间充质系列细胞(如成骨、成软骨及成脂肪细胞等)或非间充质系列细胞分化的潜能，并具有独特的细胞因子分泌功能以及免疫调节和抗炎作用的细胞，已报道可用于治疗免疫相关性疾病，如系统性红斑狼疮、克罗恩病、移植物抗宿主病；另有一些基础研究及临床试验证明间充质干细胞可减轻动脉粥样硬化。
间充质干细胞的生物活性直接影响了其治疗效果，如何在保证其生物活性的基础上实现长时间的保存和运输的便捷性已然成为现在间充质干细胞药物的关键性问题。生理盐水或葡萄糖溶液作为间充质干细胞保存液因营养缺乏，溶液等张力或等渗力等原因，导致细胞在长期保存或运输中出现活力低下、大量溶胀死亡以及细胞结团率升高等现象。含有胎牛血清或者细胞培养基的保存液因细胞接触胎牛血清时会内吞胎牛血清，进而引起细胞某些蛋白表达特性的变化，细胞回输人体后还可能引起过敏反应。而近年来人们新开发的保存液虽然较之前的保存液，改善了其细胞生物活性低下，胎牛血清使用弊端等问题，但存在配制过程复杂，细胞保存时间短，不便于运输等问题。因此成分稳定，营养物质多元化，渗透压接近血液，缓冲能力强，保存时间久便于运输的间充质干细胞保存液是目前间充质干细胞药物研发亟待需要解决的问题之一。
发明内容
本发明提供了一种细胞保存液，其特征在于，该细胞保存液包含人血清白蛋白、葡萄糖、维生素C、乳酸钠林格注射液和复方氨基酸注射液。可选地，该细胞保存液还包含人血清。
本发明还提供一种细胞制剂，其中包含本发明所述细胞保存液和保存于其中的细胞。在一些实施方式中，所述细胞为干细胞，优选为间充质干细胞，例如脐带间充质干细胞、脂肪间充质干细胞、宫膜间充质干细胞和毛囊间充质干细胞。
本发明还提供了一种试剂盒，所述试剂盒含有：
(1)本发明所述的细胞保存液，和
(2)细胞稀释液，所述细胞稀释液选自：复方氯化钠注射液或生理盐水。
本发明还提供另一种试剂盒，所述试剂盒含有：
(1)本发明所述的细胞制剂，和
(2)细胞稀释液，所述细胞稀释液选自：复方氯化钠注射液或生理盐水。
附图说明
图1显示了实施例2中毛囊间充质干细胞HF015在4℃保存120小时成脂和成骨分化能力结果图。
图2显示了实施例2中毛囊间充质干细胞HF015在4℃保存120小时细胞再贴壁能力结果图。
图3显示了实施例2中毛囊间充质干细胞HF015在4℃保存120小时流式凋亡检测结果图。
具体实施方案
本文中，如果没有特别的说明，所涉及的各组分或其优选组分可以相互组合形成新的技术方案。
本文中，如果没有特别的说明，本文所提到的所有实施方案以及优选实施方案可以相互组合形成新的技术方案。
本文中，如果没有特别的说明，本文所提到的所有技术特征以及优选特征可以相互组合形成新的技术方案。
如果没有特别指出，本说明书所用的术语“一种”指“至少一种”。
本文所公开的“范围”以下限和上限的形式。可以分别为一个或多个下限，和一个或多个上限。给定范围是通过选定一个下限和一个上限进行限定的。选定的下限和上限限定了特别范围的边界。所有可以这种方式进行限定的范围是包含和可组合的，即任何下限可以与任何上限组合形成一个范围。
具体而言，本发明首先提供了一种细胞保存液，其特征在于，它包含了人血清白蛋白(HSA)、葡萄糖、维生素C、乳酸钠林格注射液和复方氨基酸注射液的 混合物。本领域技术人员可以看出，该细胞保存液中还可以含有对细胞、对人体无害的其它组分，例如药学上可接受的载剂，例如生理盐水等稀释剂，缓冲液等pH调节剂，只要这些组分及其含量对人体无害，并且对细胞例如干细胞尤其间充质干细胞的活性、功能和稳定性没有不利的影响。
在一些实施方式中，所述细胞保存液是由人血清白蛋白、葡萄糖、维生素C、乳酸钠林格注射液和复方氨基酸注射液组成的。
在一些实施方式中，所述细胞保存液包含人血清白蛋白、葡萄糖、维生素C、人血清、乳酸钠林格注射液和复方氨基酸注射液。
在一些实施方式中，所述细胞保存液是由人血清白蛋白、葡萄糖、维生素C、人血清、乳酸钠林格注射液和复方氨基酸注射液组成的。
本发明所用的人血清白蛋白(HSA)、葡萄糖、维生素C、人血清，乳酸钠林格注射液和复方氨基酸注射液可以是从商业途径购买，也可以使用任何现有技术制备获得。
在一些实施方式中，所述细胞保存液中人血清白蛋白质量体积百分浓度为1％-8％，优选3％-7％，最优选4％-7％。
在一些实施方式中，所述细胞保存液中葡萄糖质量体积百分浓度为0.05％-2.5％，优选0.05％-1％，最优选0.125％。
在一些实施方式中，所述细胞保存液中维生素C质量体积百分浓度为0.01％-0.2％，优选0.05％-0.2％，最优选0.1％。
在一些实施方式中，所述细胞保存液中人血清体积百分浓度为10％-60％，优选10％-30％，最优选30％。
在一些实施方式中，所述细胞保存液中乳酸钠林格注射液和复方氨基酸注射液的体积比例为1:1-1:1.5，优选1:1，其可作为细胞保存本底液。
本文中，如果没有特别的说明，本文所提到的质量体积百分浓度(m/v)是指每百毫升(100mL)溶液中指定组分的质量克数(g)。例如，葡萄糖质量体积百分浓度(m/v)为0.05％-2.5％是指100毫升细胞保存液中含有葡萄糖粉末质量为0.05g-2.5g。所述体积百分浓度(v/v)是指单位体积溶液中指定组分体积所占的百分数，例如人血清体积百分浓度(v/v)为10％-60％是指100毫升细胞保存液中含有人血清液体体积为10mL-60mL。
在一些实施方式中，所述细胞保存液的pH为5.5-7.5，优选pH为6.0-6.5。
在一些实施方式中，所述细胞保存液的渗透压为240-360mOsmol/kg(毫渗透压摩尔浓度)；优选280-320mOsmol/kg(毫渗透压摩尔浓度)。在本发明中，可以 采用例如注射用水调节细胞保存液的离子浓度/强度，从而调节其渗透压。
本发明所用“乳酸钠林格注射液”符合药典的规定。在一些实施方式中，所述乳酸钠林格注射液包含乳酸钠，氯化钠，氯化钾，氯化钙。在一些实施方式中，所述乳酸钠林格注射液每1000mL中含有组分：乳酸钠3.10g，氯化钠6.00g，氯化钾0.30g，氯化钙0.20g。其中，氯化钙优选CaCl 2·2H 2O。
本发明所用“复方氨基酸注射液”符合药典的规定。在一些实施方式中，所述复方氨基酸注射液包含L-脯氨酸，L-丝氨酸，L-丙氨酸，L-异亮氨酸，L-亮氨酸，L-门冬氨酸，L-酪氨酸，L-谷氨酸，L-苯丙氨酸，L-精氨酸盐酸盐，L-赖氨酸盐酸盐，L-缬氨酸，L-苏氨酸，L-组氨酸盐酸盐，L-色氨酸，L-蛋氨酸，L-胱氨酸，甘氨酸，山梨醇，亚硫酸氢钠。在一些实施方式中，所述复方氨基酸注射液每1000mL含有组分：L-脯氨酸1.00g，L-丝氨酸1.00g，L-丙氨酸2.00g，L-异亮氨酸3.52g，L-亮氨酸4.90g，L-门冬氨酸2.50g，L-酪氨酸0.25g，L-谷氨酸0.75g，L-苯丙氨酸5.33g，L-精氨酸盐酸盐5.00g，L-赖氨酸盐酸盐4.30g，L-缬氨酸3.60g，L-苏氨酸2.50g，L-组氨酸盐酸盐2.50g，L-色氨酸0.90g，L-蛋氨酸2.25g，L-胱氨酸0.10g，甘氨酸7.60g，山梨醇50.00g，亚硫酸氢钠0.5g。本发明的细胞保存液可用于保存各类细胞，尤其适合用于保存干细胞，特别是间充质干细胞，例如脐带间充质干细胞、脂肪间充质干细胞、宫膜间充质干细胞和毛囊间充质干细胞。
因此，本发明还提供了一种细胞制剂，其中包含本发明的细胞保存液和保存于其中的细胞。在一些实施方式中，所述细胞为干细胞，优选为间充质干细胞，例如脐带间充质干细胞、脂肪间充质干细胞、宫膜间充质干细胞和毛囊间充质干细胞。
一些实施方式中，所述细胞制剂中的细胞密度为5×10 5-2×10 7个/mL，更优选2×10 6-2×10 7个/mL，可根据临床需求调整。
本发明中，干细胞制剂的保存温度以4℃以下为宜，优选0-4℃，保存时间优选1-7天，例如1-5天。
在一些实施方式中，间充质干细胞在本发明提供的细胞保存液中保存120小时，甚至180小时后，其活率仍然能够保持在80％以上，更好的，可达90％以上。本文中，细胞活率是指活细胞数占总体细胞数的比率。
在一些实施方式中，所述细胞保存液的制备方法包括如下步骤：
1)将乳酸钠林格注射液和复方氨基酸注射液(优选体积比例为1:1-1:1.5，更优选1:1)混合均匀；
2)将人血清白蛋白(优选质量体积百分浓度为1％-8％，更优选3％-7％，最优选4％-7％)，葡萄糖(优选质量体积百分浓度为0.05％-2.5％，更优选0.05％-1％， 最优选0.125％)，维生素C(优选质量体积百分浓度为0.01％-0.2％，更优选0.05％-0.2％，最优选0.1％)，可选的人血清(如有，优选体积百分浓度为10％-60％，更优选10％-30％，最优选30％)加入到步骤1)得到的溶液中混合均匀。
细胞制剂直接应用于人体时，可能需要在临用前根据临床需要稀释细胞制剂并且复温。因此，本发明还公开了一种试剂盒，所述试剂盒含有：
(1)本发明所公开的细胞保存液，和
(2)细胞稀释液，所述细胞稀释液选自：复方氯化钠注射液或生理盐水。
本发明还提供另一种试剂盒，所述试剂盒含有
(1)本发明所述的细胞制剂，和
(2)细胞稀释液，所述细胞稀释液选自：复方氯化钠注射液或生理盐水。
此处，用作细胞稀释剂的复方氯化钠注射液包含氯化钠，氯化钾，氯化钙。在一些实施方式中，复方氯化钠注射液每1000mL中含有氯化钠8.50g，氯化钾0.30g，氯化钙0.33g。生理盐水为生理学实验或临床上常用的渗透压与动物或人体血浆的渗透压基本相等的氯化钠溶液，例如0.9％(w/v)氯化钠溶液。
在一些实施方式中，所述试剂盒还包含使用说明，教导用细胞稀释液按照一定的稀释比例稀释本发明的细胞制剂或是用本发明细胞保存液配制的细胞制剂。例如，稀释比例可以是1:40-1:5，优选1:20-1:10。可选地，使用说明还教导将稀释后的细胞保存液放置于室温复温之后回输人体，放置时间为1-24小时，优选1-10小时，更优选5小时。
下面将结合实施例进一步详细地描述本发明。然而应当理解，列举这些实施例只是为了起说明作用，而并不是用来限制本发明的范围。
实施例
实施例中所用的间充质干细胞的制备方法如下所述：脐带间充质干细胞制备方法参照文献：韩华,薛改,张俊勤，等.“人脐带间充质干细胞的分离、培养及鉴定”[J].河北医科大学学报,2015,36(1):21-23；宫膜间充质干细胞制备方法参照文献：周云帆,杨波,胡祥,等.“经血源性子宫内膜间充质干细胞的分离、培养与鉴定”[J].中国组织工程研究,2010,14(32):5952-5956；脂肪间充质干细胞制备方法参照文献：赵璞,丁幸坡.“人脂肪干细胞的分离培养与鉴定”[J].生物医学工程与临床,2017(04):13-18；毛囊间充质干细胞制备方法参照文献：杨丽.“人毛囊源性间充质样干细胞的分离培养与成骨诱导”[D].吉林大学,2010。
实施例1：不同配方细胞保存液保存间充质干细胞的活率检测
1)取乳酸钠林格注射液(购买自石家庄四药有限公司)和复方氨基酸注射液(购买自石家庄四药有限公司)，分别按照体积比1:1和1:1.5进行混匀，即为细胞保存液本底液。
2)按照表1不同配方的比例配制细胞保存液。配制方法为先将人血清白蛋白、葡萄糖和维生素C粉末添加到步骤1)本底液中混匀，再加入人血清(AB型，购买自上海恒远生物科技有限公司)，用步骤1)配制得到的本底液定容到1L，混合均匀后，用20mL注射器吸取后用0.22μm的滤器进行过滤除菌。配制出了4个不同配方的细胞保存液(表1)。
3)取离心好的脐带、毛囊、宫膜和脂肪间充质干细胞用上述配制好的不同配方的细胞保存液进行重悬，最终的细胞密度为2×10 6-5×10 6个/mL。
制得的细胞制剂置于4℃冰箱内低温保存。保存180小时后，采用AO/PI(AO(Acridine Orange)吖啶橙，PI(Propidium Iodide)碘化丙啶)双染细胞凋亡检测试剂盒(DNA探针双染细胞核方法，购买自上海睿钰生物科技有限公司)对不同配方的细胞保存液保存的间充质干细胞进行细胞活率检测，结果见表2。
表1细胞保存液配方表
配方编号 人血白蛋白/g 维生素C/g 葡萄糖/g 人血清/ml 本底液比例 1
1 30.00 1.057 1.25 300 1:1
2 10.00 0.704 2.5 100 1:1.5
3 40.00 1.408 0.625 300 1:1
4 50.00 0.176 2.5 600 1:1
注：1.该比例指本底液中乳酸钠林格注射液:复方氨基酸注射液体积比
表2不同配方的细胞保存液保存180小时后细胞活率检测结果
Figure PCTCN2021083169-appb-000001
实验结论：通过检测本发明不同范围的配方细胞保存液保存的间充质干细胞的细胞活率，发现本发明的细胞保存液对于脐带、脂肪、毛囊和宫膜间充质干细胞均具有良好的保存能力，保存180小时后四种细胞的细胞活率仍能维持在80％以上，部分培养细胞活率甚至超过90％。
实施例2：毛囊间充质干细胞保存于细胞保存液120小时后细胞各性能检测
1、细胞活率检测
分别取按照实施例1所述保存于配方1细胞保存液0小时，96小时，120小时后的毛囊间充质干细胞(编号为“HF015”)测试细胞活率。采用AO/PI双染细胞凋亡检测试剂盒进行检测，结果见表3。
2、细胞分化能力测试
分别取按照实施例1所述保存于配方1细胞保存液0小时，96小时，120小时后的毛囊间充质干细胞HF015，以细胞浓度为1.0×10 5个/孔，分别将不同保存时间的毛囊间充质干细胞均匀铺到包被好的十二孔板中，置于5％CO 2、37℃培养箱中培养3-4天至细胞融合率达到90％-100％，于超净台中吸弃孔内培养基，分别加入1ml成骨诱导分化完全培养基(购买自赛业生物科技有限公司)，每3天更换一次培养基，成骨诱导分化21天时，用茜素红S(购买自北京索莱宝科技有限公司)对成骨分化细胞进行染色。结果见表3和图1。
分别取保存于实施例1中配方1细胞保存液0小时，96小时，120小时后的毛囊间充质干细胞HF015，以细胞浓度为1.0×10 5个/孔，分别将不同保存时间的毛囊间充质干细胞均匀铺到包被好的十二孔板中，置于5％CO 2、37℃培养箱中培养3-4天至细胞融合率90％-100％左右，于超净台中吸弃孔内培养基，分别加入1ml成脂诱导分化培养基(购买自STEMCELL)，每3天更换一次培养基，成脂诱导分化14天时，用油红O染色(购买自北京索莱宝科技有限公司)对成脂分化细胞染色。结果见表3和图1。
3、细胞再贴壁能力测试
分别取保存于实施例1中配方1细胞保存液0小时，96小时，120小时后的毛囊间充质干细胞HF015，按照细胞密度为10000个/cm 2进行复苏接种，接种24小时后镜下评估细胞融合度。结果见表3和图2。
4、细胞凋亡检测
分别取保存于实施例1中配方1细胞保存液0小时，96小时，120小时后的毛囊间充质干细胞HF015，获得重悬细胞，按照“Annexin V-APC/PI细胞凋亡检测试剂盒”(购买自联科生物公司)的说明书操作，上流式细胞分析仪进行检测，结果见表3和图3。“Annexin”即膜联蛋白，“PI”即碘化丙啶。
5、实验结果分析
细胞各性能检测结果见表3。
表3细胞各性能检测结果
Figure PCTCN2021083169-appb-000002
实验结论：细胞保存液中的毛囊间充质干细胞在4℃保存120小时后，细胞活率大于90％，流式凋亡检测活率大于90％，细胞再贴壁能力大于20％；与0小时相比，低温放置96小时和120小时后细胞成脂和成骨分化能力无显著变化。故毛囊间充质干细胞在该细胞保存液中，4℃可稳定保存120小时，不影响细胞活率及分化潜能。
实施例3：保存于不同浓度葡萄糖的细胞保存液中的间充质干细胞活率检测
1)按照表4的配方，配制方法同实施例1，配制七个不同浓度葡萄糖的细胞保存液。
2)取离心好的脐带和脂肪间充质干细胞用上述配制好的不同葡萄糖浓度的细胞保存液进行重悬，最终的细胞密度为2×10 6-5×10 6个/mL。
制得的细胞制剂置于4℃冰箱内低温保存。保存180小时后，采用AO/PI双染细胞凋亡检测试剂盒对不同浓度葡萄糖配方的细胞保存液保存的间充质干细胞进行细胞活率检测，结果见表5。
表4细胞保存液配方表
Figure PCTCN2021083169-appb-000003
注：1.该比例指本底液中乳酸钠林格注射液:复方氨基酸注射液体积比
表5不同浓度葡萄糖的细胞保存液保存180小时后细胞活率检测结果
Figure PCTCN2021083169-appb-000004
Figure PCTCN2021083169-appb-000005
实验结论：通过检测不同浓度葡萄糖细胞保存液保存的脐带和脂肪间充质干细胞的细胞活率，发现高浓度的葡萄糖(例如质量体积百分比浓度为4％及以上)对于维持脐带和脂肪间充质干细胞的细胞活率不具有优势，均低于70％，而低浓度的葡萄糖(例如质量体积百分比浓度为0.0625％-2.5％)有利于维持细胞活率，其可以维持细胞活率在80％以上。
实施例4：不同稀释制剂稀释的细胞保存液室温保存5小时后细胞活率检测
实验方法：间充质干细胞如实施例1所述配制于配方1细胞保存液中，4℃保存24小时或48小时后，按照1：10用稀释液(如乳酸钠林格注射液、复方氨基酸注射液、生理盐水或5％葡萄糖注射液)稀释，室温放置5小时，采用AO/PI双染细胞凋亡检测试剂盒检测细胞活率，检测结果见表6。
表6不同稀释制剂稀释的细胞保存液室温保存5小时细胞活率检测结果
Figure PCTCN2021083169-appb-000006
实验结论：采用不同的稀释制剂复温，对于同一种来源的间充质干细胞的活率影响不同；并且，采用同一种稀释制剂复温，对于不同来源的间充质干细胞的活率影响也不同。采用5％的葡萄糖注射液作为稀释液在室温下放置5小时后，四种不同来源的间充质干细胞的细胞活率均下降到90％以下；采用乳酸钠林格注射液作为稀释液在室温下放置5小时后，脂肪间充质干细胞活率下降到80％以下；而复方氨基酸注射液、生理盐水和复方氯化钠注射液作为回输稀释液可以在室温条件下维持不同来源的间充质干细胞的活率，5小时内细胞活率可维持在90％以上，较其他两种制剂具有明显优势。
然而，采用复方氨基酸注射液作为稀释液时，会造成细胞保存液渗透压过高，不利于细胞的保存。因此，本发明选用生理盐水或复方氯化钠注射液作为稀释液，制备细胞保存液。
实施例5：保存于不同浓度人血白蛋白的无血清细胞保存液中的间充质干细胞活率检测
1)取乳酸钠林格注射液和复方氨基酸注射液按照体积比1:1进行混匀，即为细胞保存液本底液。
2)按照表7不同配方配制细胞保存液。配制方法为将人血清白蛋白、葡萄糖和维生素C粉末依次添加到步骤1)本底液中混匀，再加入人血清(如有)，用步骤1)配制得到的本底液和注射水按体积比为2：3混合所得溶液定容到1L，混合均匀后，用20mL注射器吸取后用0.22μm的滤器进行过滤除菌。配制出了6个不同配方的细胞保存液，并且测量其pH值和渗透压。(表7)。
表7不同浓度人血白蛋白的细胞保存液配方表
Figure PCTCN2021083169-appb-000007
取离心好的毛囊和宫膜间充质干细胞用上述配制好的不同配方的细胞保存液进行重悬，最终的细胞密度为2×10 6-5×10 6个/mL。制得的细胞制剂置于4℃冰箱内低温保存。保存144小时后，采用Annexin V-FITC/PI细胞凋亡试剂盒(杭州联科生物技术股份有限公司)对不同配方的细胞保存液保存的毛囊间充质干细胞进行细胞活率检测，结果见表8。
表8不同浓度人血白蛋白的细胞保存液保存144小时细胞活率检测结果
Figure PCTCN2021083169-appb-000008
Figure PCTCN2021083169-appb-000009
实验结论：通过检测上述宫膜间充质干细胞和毛囊间充质干细胞的细胞活率，发现上述不同浓度人血白蛋白的细胞保存液对于两种间充质干细胞均具有良好的保存能力。除配方2，保存144小时后两种间充质干细胞的细胞活率基本能维持在90％以上。具体而言，与配方1相比，4％-7％质量体积百分浓度人血白蛋白的无血清细胞保存液同样能很好地维持间充质干细胞的活率；并且，无血清细胞保存液中，人血白蛋白的质量体积百分浓度优选5％。

Claims (11)

  1. 一种细胞保存液，其特征在于，所述细胞保存液包含人血清白蛋白、葡萄糖、维生素C、乳酸钠林格注射液和复方氨基酸注射液；优选地，所述细胞保存液由人血清白蛋白、葡萄糖、维生素C、乳酸钠林格注射液和复方氨基酸注射液组成。
  2. 根据权利要求1所述的细胞保存液，其特征在于，所述细胞保存液还包含人血清；优选地，所述细胞保存液由人血清白蛋白、葡萄糖、维生素C、乳酸钠林格注射液、复方氨基酸注射液和人血清组成。
  3. 根据权利要求1或2所述的细胞保存液，其特征还在于以下一项或多项：
    1)所述细胞保存液中人血清白蛋白质量体积百分浓度为1％-8％，优选3％-7％，更优选4％-7％；
    2)所述细胞保存液中葡萄糖质量体积百分浓度为0.05％-2.5％，优选0.05％-1％，最优选0.125％；
    3)所述细胞保存液中维生素C质量体积百分浓度为0.01％-0.2％，优选0.05％-0.2％，最优选0.1％；
    4)所述细胞保存液中人血清体积百分浓度为10％-60％，优选10％-30％，最优选30％；
    5)所述细胞保存液中乳酸钠林格注射液和复方氨基酸注射液的体积比例为1:1-1:1.5，优选1:1。
  4. 根据权利要求1或2所述的细胞保存液，其特征在于，所述乳酸钠林格注射液包含乳酸钠，氯化钠，氯化钾，氯化钙；
    优选地，所述乳酸钠林格注射液每1000mL中含有乳酸钠3.10g，氯化钠6.00g，氯化钾0.30g，氯化钙0.20g。
  5. 根据权利要求1或2所述的细胞保存液，其特征在于，所述复方氨基酸注射液包含L-脯氨酸，L-丝氨酸，L-丙氨酸，L-异亮氨酸，L-亮氨酸，L-门冬氨酸，L-酪氨酸，L-谷氨酸，L-苯丙氨酸，L-精氨酸盐酸盐，L-赖氨酸盐酸盐，L-缬氨酸，L-苏氨酸，L-组氨酸盐酸盐，L-色氨酸，L-蛋氨酸，L-胱氨酸，甘氨酸，山梨醇，亚硫酸氢钠；
    优选地，所述复方氨基酸注射液每1000mL含有L-脯氨酸1.00g，L-丝氨酸1.00g，L-丙氨酸2.00g，L-异亮氨酸3.52g，L-亮氨酸4.90g，L-门冬氨酸2.50g，L-酪氨酸0.25g，L-谷氨酸0.75g，L-苯丙氨酸5.33g，L-精氨酸盐酸盐5.00g，L-赖氨酸盐酸盐4.30g，L-缬氨酸3.60g，L-苏氨酸2.50g，L-组氨酸盐酸盐2.50g，L-色氨酸0.90g， L-蛋氨酸2.25g，L-胱氨酸0.10g，甘氨酸7.60g，山梨醇50.00g，亚硫酸氢钠0.5g。
  6. 根据权利要求1或2所述的细胞保存液，其特征在于，所述细胞保存液的渗透压为240-360mOsmol/kg，优选280-320mOsmol/kg；
    和/或，所述细胞保存液的pH为5.5-7.5，优选pH为6.0-6.5。
  7. 一种细胞制剂，其特征在于，包含细胞和权利要求1-6中任一项所述的细胞保存液；优选地，所述细胞为干细胞；更优选地，所述干细胞为间充质干细胞，例如脐带间充质干细胞、脂肪间充质干细胞、宫膜间充质干细胞或毛囊间充质干细胞。
  8. 根据权利要求7所述的细胞制剂，其中，细胞密度为5×10 5-2×10 7个/mL，优选2×10 6-2×10 7个/mL。
  9. 一种试剂盒，其特征在于，所述试剂盒含有：
    1)权利要求1-6中任一项所述的细胞保存液，和
    2)细胞稀释液，所述细胞稀释液选自：复方氯化钠注射液或生理盐水。
  10. 一种试剂盒，其特征在于，所述试剂盒含有：
    1)权利要求7或8所述的细胞制剂，和
    2)细胞稀释液，所述细胞稀释液选自：复方氯化钠注射液或生理盐水；
  11. 如权利要求9或10所述的试剂盒，所述复方氯化钠注射液包含氯化钠，氯化钾，氯化钙；
    优选地，所述复方氯化钠注射液每1000mL中含有氯化钠8.50g，氯化钾0.30g，氯化钙0.33g。
PCT/CN2021/083169 2020-03-27 2021-03-26 细胞保存液及其试剂盒 WO2021190620A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202180023404.0A CN115315182A (zh) 2020-03-27 2021-03-26 细胞保存液及其试剂盒

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010231327.5 2020-03-27
CN202010231327 2020-03-27

Publications (1)

Publication Number Publication Date
WO2021190620A1 true WO2021190620A1 (zh) 2021-09-30

Family

ID=77890178

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/083169 WO2021190620A1 (zh) 2020-03-27 2021-03-26 细胞保存液及其试剂盒

Country Status (2)

Country Link
CN (1) CN115315182A (zh)
WO (1) WO2021190620A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114052008A (zh) * 2021-11-23 2022-02-18 中山大学附属第三医院（中山大学肝脏病医院） 一种保存液及其应用

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103783031A (zh) * 2012-10-29 2014-05-14 四川新生命干细胞科技股份有限公司 一种细胞保存液
CN104542578A (zh) * 2015-02-05 2015-04-29 广州赛莱拉干细胞科技股份有限公司 一种细胞保存液及其制备方法、应用
CN104920340A (zh) * 2015-07-15 2015-09-23 广州赛莱拉干细胞科技股份有限公司 一种免疫细胞保存液及其应用
CN106386782A (zh) * 2016-06-24 2017-02-15 安徽未名细胞治疗有限公司 一种脐带保存液
CN107347871A (zh) * 2017-06-07 2017-11-17 江苏善之源健康科技有限公司 一种免疫细胞回输保存液
WO2018031922A1 (en) * 2016-08-11 2018-02-15 Sulfilatec, Inc. Medical and nutritional compositions and methods of use
WO2018187439A1 (en) * 2017-04-06 2018-10-11 The University Of North Carolina At Chapel Hill Cryopreservation method
CN108651442A (zh) * 2018-05-17 2018-10-16 广东芙金干细胞再生医学有限公司 一种间充质干细胞4℃储存液
CN109329270A (zh) * 2018-11-19 2019-02-15 成都清科生物科技有限公司 一种静脉回输用间充质干细胞保存液及其制备方法
CN110100812A (zh) * 2019-05-30 2019-08-09 南京艾德免疫治疗研究院有限公司 一种免疫细胞的冻存回输液、冻存方法及回输应用
CN110367242A (zh) * 2019-08-02 2019-10-25 陕西佰傲干细胞再生医学有限公司 凋亡小体保存液及凋亡小体的保存方法
CN110622956A (zh) * 2019-09-27 2019-12-31 广州南医大生物工程有限公司 一种脐带间充质干细胞保存液

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103404509B (zh) * 2013-08-07 2015-04-08 彭乐 一种细胞保存液、其制备方法和应用
CN108244097B (zh) * 2018-01-10 2018-11-09 暨赛再生医学科技有限公司 一种长期保存过表达bFGF的C17.2的神经干细胞保存液
CN109845725A (zh) * 2019-01-25 2019-06-07 北京益华生物科技有限公司 细胞运输保存液及其应用

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103783031A (zh) * 2012-10-29 2014-05-14 四川新生命干细胞科技股份有限公司 一种细胞保存液
CN104542578A (zh) * 2015-02-05 2015-04-29 广州赛莱拉干细胞科技股份有限公司 一种细胞保存液及其制备方法、应用
CN104920340A (zh) * 2015-07-15 2015-09-23 广州赛莱拉干细胞科技股份有限公司 一种免疫细胞保存液及其应用
CN106386782A (zh) * 2016-06-24 2017-02-15 安徽未名细胞治疗有限公司 一种脐带保存液
WO2018031922A1 (en) * 2016-08-11 2018-02-15 Sulfilatec, Inc. Medical and nutritional compositions and methods of use
WO2018187439A1 (en) * 2017-04-06 2018-10-11 The University Of North Carolina At Chapel Hill Cryopreservation method
CN107347871A (zh) * 2017-06-07 2017-11-17 江苏善之源健康科技有限公司 一种免疫细胞回输保存液
CN108651442A (zh) * 2018-05-17 2018-10-16 广东芙金干细胞再生医学有限公司 一种间充质干细胞4℃储存液
CN109329270A (zh) * 2018-11-19 2019-02-15 成都清科生物科技有限公司 一种静脉回输用间充质干细胞保存液及其制备方法
CN110100812A (zh) * 2019-05-30 2019-08-09 南京艾德免疫治疗研究院有限公司 一种免疫细胞的冻存回输液、冻存方法及回输应用
CN110367242A (zh) * 2019-08-02 2019-10-25 陕西佰傲干细胞再生医学有限公司 凋亡小体保存液及凋亡小体的保存方法
CN110622956A (zh) * 2019-09-27 2019-12-31 广州南医大生物工程有限公司 一种脐带间充质干细胞保存液

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114052008A (zh) * 2021-11-23 2022-02-18 中山大学附属第三医院（中山大学肝脏病医院） 一种保存液及其应用

Also Published As

Publication number Publication date
CN115315182A (zh) 2022-11-08

Similar Documents

Publication Publication Date Title
JP6850360B2 (ja) 臨床使用可能な細胞凍結保存液
TWI640629B (zh) Mammalian cell transplantation solution containing trehalose and polyglucose
US20200170242A1 (en) Cell cryopreservation formulation and cell recovery method
WO2012048093A2 (en) Enhanced msc preparations
CN111248191A (zh) 一种常温细胞保存液和注射用细胞制剂
CN114557337B (zh) 脐带间充质干细胞无蛋白非程序冻存液及其制备方法
CN104839146A (zh) 组合物及其用途、胎盘保存制剂及其制备方法
TW201720922A (zh) 細胞擴增方法及治療組成物
JPWO2016076428A1 (ja) 臍帯血および末梢血の凍結保存方法および凍結保存用溶液
WO2021190620A1 (zh) 细胞保存液及其试剂盒
CN116458493A (zh) 一种无dmso的干细胞保存液及其制备方法和使用方法
WO2024131428A1 (zh) 干细胞制剂组合物及其批量生产方法和临床应用
CN105039251B (zh) 一种pH值稳定的淋巴细胞无血清培养基及其制备方法和应用
CN113973805A (zh) 细胞冻存试剂盒及其使用方法
WO2021056885A1 (zh) 一种用于人月经血的保存液及其制备方法
CN115786247B (zh) 一种无血清培养基及其在维持毛囊活性、毛发养护及移植方面的应用
CN114391536B (zh) 一种间充质干细胞冻存液
US20230014549A1 (en) Mesenchymal stem cells for use in the treatment of chronic kidney disease
CN107787960B (zh) 视网膜色素上皮细胞的冻存液及其应用
CN115323498A (zh) 一种即用型临床级脐带间充质干细胞工作库的构建方法及应用
WO2021092564A1 (en) Cultivated autologous limbal epithelial cell (calec) transplantation
RU2303631C1 (ru) Среда для криоконсервирования мезенхимальных стволовых клеток и биотрансплантат с ее использованием
CN110295140A (zh) 一种无血清培养骨髓间充质干细胞的方法
CN116396930B (zh) 间充质干细胞无血清培养基及其应用
CN115281184B (zh) 一种间充质干细胞复合冻存液及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21775251

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21775251

Country of ref document: EP

Kind code of ref document: A1