WO2018064950A1 - 一种保持冻存细胞活性的干细胞凝胶制剂及其应用 - Google Patents

一种保持冻存细胞活性的干细胞凝胶制剂及其应用 Download PDF

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WO2018064950A1
WO2018064950A1 PCT/CN2017/103948 CN2017103948W WO2018064950A1 WO 2018064950 A1 WO2018064950 A1 WO 2018064950A1 CN 2017103948 W CN2017103948 W CN 2017103948W WO 2018064950 A1 WO2018064950 A1 WO 2018064950A1
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gel preparation
cell
stem cell
gel
sodium alginate
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French (fr)
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耿洁
梁璐
韩忠朝
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北京汉氏联合干细胞研究院有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a gel preparation for cell cryopreservation;
  • the present invention also relates to a stem cell gel preparation comprising mesenchymal stem cells and the above gel preparation;
  • the invention also relates to the use of the above stem cell gel formulation.
  • Tissue engineering scaffold material should be a permanent substitute and must be a biodegradable material.
  • the material itself should be non-immunogenic to the tissue, and the product of the material degradation should have no toxicity or abnormal reaction to the biological tissue.
  • collagen has good affinity to cells, it is often derived from animal tissues. The risk of pathogenic microorganisms is high, and it is immunogenic, and the price is high, and the mechanical properties are poor. It needs to be used after cross-linking.
  • Conventional treatment is to disinfect, expose or bandage the external application on the wound surface, but the effect is not ideal. The lighter patients feel painful, red and swollen wounds, severe empyema under the armpits, limited limb activity, and then affect normal work.
  • Sodium alginate is a linear anionic polysaccharide extracted from the brown algae of the ocean. It has good biocompatibility, biodegradability and immunoisolation properties, and is the most commonly used bioscaffold material.
  • the sodium alginate aqueous solution is a light yellow semi-fluid gelatinous liquid, which can quickly and evenly cover the wound surface, slow the evaporation of surface moisture, maintain cell activity, protect and support cells, and is a good carrier for extracellular treatment of skin tissue repair. It can also be used as a cell scaffold material for intestinal tissue repair and uterine tissue repair.
  • Mesenchymal cells are one of the best seed cells in tissue engineering because of their easy separation, culture and amplification. They can maintain their dryness in vitro and have a unique genetic background. Mesenchymal stem cells have low immunogenicity and will not cause abnormal immune response in the body; they can release a variety of anti-inflammatory factors, inhibit the body's inflammatory response, and release a variety of factors that promote tissue damage repair to promote tissue damage repair; mobilize the body's own Stem cells are involved in tissue damage repair, and direct contact with defects or inflammatory wounds can maximize the repair and treatment of stem cells.
  • cryopreservation of liquid nitrogen which can maintain cell viability of more than 80%.
  • liquid nitrogen is not easily transported by vehicles, and cell liquid nitrogen freezing is greatly restricted in the case of transfer transportation.
  • the application location needs Equipped with liquid nitrogen storage and liquid nitrogen supply, subject to local geography and conditions.
  • Chinese patent application CN 101451124A discloses a preparation method of a human umbilical cord mesenchymal stem cell wound spreader using methyl cellulose as a cell matrix, which comprises an antibiotic gentamicin, is not suitable for antibiotic allergy population; and the spreader is fast Formulated with fetal-derived fetal bovine serum, it may cause allergies and animal-borne safety hazards; there are many operational steps in the rapid preparation, complicated process, and the risk of microbial contamination. It can not be completed for 2 weeks according to the requirements of the Pharmacopoeia before transportation. Inspection, product quality can not be guaranteed.
  • Chinese patent application CN 102670654A discloses a stem cell preparation for wound repair comprising mesenchymal stem cells derived from umbilical cord, placenta or amniotic membrane, and a polymer stabilizer selected from the group consisting of sodium alginate and transparent Sodium carbonate, chitosan, hydroxyethyl starch may also contain cell antifreeze DMSO, humectant propylene glycol or glycerol.
  • the preparation has a good therapeutic effect, it has certain disadvantages in terms of stability and cell survival rate.
  • Another object of the present invention is to provide a stem cell gel preparation containing mesenchymal stem cells and the gel preparation.
  • a gel formulation for maintaining the activity of cryopreserved cells comprises the following mass percentage components: 1-3% sodium alginate, 1-5% dimethyl sulfoxide, 1- 5% propylene glycol, 1-5% polyphenols, 2%-10% dextran, 1-5% human albumin, make up water or phosphate solution.
  • the phosphate solution component is 0.02% potassium chloride, 0.0047% magnesium chloride, 0.1158% disodium hydrogen phosphate, 0.020% sodium dihydrogen phosphate, 0.8% sodium chloride and water for injection.
  • a preferred composition is: 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 2% polyphenol, 6% dextran and 2% human albumin, supplemented with a phosphate solution.
  • the phosphate solution component is 0.02% potassium chloride, 0.0047% magnesium chloride, 0.1158% disodium hydrogen phosphate, 0.020% sodium dihydrogen phosphate, 0.8% sodium chloride and water for injection.
  • the gel preparation of the invention has the composition of polyphenols, dextran and human albumin, and the polyphenol has very strong antioxidant activity, and the antioxidant activity is at least More than 100 times that of vitamin C is 25 times that of vitamin E, which protects cells and DNA from damage.
  • Dextran is currently one of the best plasma substitutes, which can greatly increase cell viability and reduce the amount of DMSO used.
  • human serum albumin renders the gel formulation of the present invention free of animal-derived ingredients, improving the safety and stability of the formulation. Further, the composition ratio of each of the gel preparations was screened to form a gel preparation suitable for maintaining the activity of the frozen cells.
  • the gel preparation of the present invention can be used for cryopreservation of various cells including, but not limited to, tumor cell lines, animal cells, stem cells, and the like of various origins.
  • a stem cell gel preparation comprising mesenchymal stem cells and the gel preparation, preferably containing 0.5 to 3 ⁇ 10 6 mesenchymal stem cells per 1 ml of the gel preparation.
  • the mesenchymal stem cells may be derived from the umbilical cord, the placenta, and the amniotic membrane tissue, and are prepared by taking the above-mentioned tissue, but are not limited thereto, and may be mesenchymal stem cells of any origin.
  • the stem cell gel preparation of the invention is prepared as follows: the sterile sodium alginate powder is dissolved in a phosphate solution, swelled at room temperature for 24-48 hours, thoroughly stirred and mixed, and then added to the remaining group to be formulated into 1- 3% sodium alginate gel preparation. After standing, mesenchymal stem cells were added and uniformly mixed to prepare a stem cell gel preparation.
  • the optimum mesh size of the sodium alginate to prepare the gel preparation is 200, and the viscosity is 600-1000 cps.
  • the pH of the solution is 6.5-7.5.
  • the cell-rich gel preparation can be directly stored at -80 ° C to -60 ° C for long-term storage, and the cryopreservation method is non-program cooling. The shelf life is up to one and a half years. After resuscitating the cell-rich gel preparation, it can be used directly, and the cell viability can be maintained above 90%. If not used immediately, it can also be stored at 4 ° C, and the cell viability can be maintained above 80% within 6 hours.
  • the stem cell gel preparation of the present invention for preparing a medicament for treating skin damage or mucosal damage, and preparing a stem cell gel preparation comprising the gel preparation of the present invention and mesenchymal stem cells.
  • the skin lesions or mucosal lesions include, but are not limited to, skin ulcers, acne, diabetic foot, ulcerative colitis, Crohn's disease, endometrial damage.
  • the gel preparation of the invention has good biocompatibility, can maintain high activity of cells, and does not require liquid nitrogen freezing.
  • the cells can be stored at a low temperature for easy transportation and convenient use.
  • the thawing method is simple and rapid, and does not require traditional methods such as washing, centrifugation, etc., reducing the influence of human operation on cell viability and yield and the risk of contamination. After thawing, the cell gel preparation after resuscitation can still be maintained. High activity of cells and dryness of cells.
  • Figure 1 Effect of different sodium alginate materials on cell viability
  • Figure 7 Comparison of the gel formulation of the gel formulation of the present invention and the control group on the ordinate of the cell viability
  • Figure 8 Activity of mesenchymal stem cells from different sources in sodium alginate gel
  • Figure 9 Cell viability of the gel preparation of the present invention after long-term cryopreservation
  • Figure 10 Therapeutic effect of the stem cell gel preparation of the present invention on rat skin lesions.
  • Mesenchymal stem cells are derived from, but not limited to, umbilical cord, placenta, and amniotic tissue donated by healthy people, obtained by tissue isolation, culture amplification, and passage.
  • mesenchymal stem cells were routinely cultured in DMEM medium (complete medium) containing 10% fetal bovine serum at 37 ° C under 5% CO 2 until the cell fusion degree reached 90%. When left and right, digested with 0.25% trypsin, and then stopped with the above complete medium, the obtained cell suspension was centrifuged, the supernatant was discarded, and the cell pellet was suspended in serum-free DMEM medium to adjust the cell suspension.
  • the content of mesenchymal stem cells is 0.4 ⁇ 10 6 to 2.5 ⁇ 10 7 /ml.
  • cell viability (%) total number of viable cells / (total number of viable cells + total number of dead cells) ⁇ 100%
  • cell yield (%) total number of live cells at the test point / total number of initial viable cells ⁇ 100%
  • 200 mesh, 100 mesh and 50 mesh sodium alginate powder were prepared into 1%, 2%, 3% sodium alginate solution, added 3% dimethyl sulfoxide, 5% propylene glycol, and then added after cryopreservation
  • Mesenchymal stem cells were mixed, uniformly mixed, placed in a refrigerator at 4 ° C, and cell viability and cell yield were compared at 0, 1, and 6 hours. It can be seen from the experimental results (Fig. 1) that 200-mesh sodium alginate is the most suitable gel material for cell survival.
  • the phosphate solution was composed of 0.02% potassium chloride, 0.0047% magnesium chloride, 0.1158% disodium hydrogen phosphate, 0.020% sodium dihydrogen phosphate, 0.8% sodium chloride and water for injection.
  • the clarity of 1%, 2% and 3% gels is very good, and 4% of the gels have insolubles.
  • the fluidity of the gel decreases with increasing sodium alginate concentration, and the 3% gel has the least fluidity and the best adhesion. Based on the results of comprehensive clarity and fluidity, the final concentration of sodium alginate was chosen as 3%.
  • the experiment is divided into 4 groups:
  • the components of each group were thoroughly mixed to prepare a stem cell gel preparation.
  • the stem cell gel preparation was taken as a -1 day count before cryopreservation, and three sets of stem cell gel preparations of each group were taken at 1 hour, 6 hours, and 24 hours after cryopreservation for cell viability and cell yield detection. It can be seen from the experimental results (Fig. 2) that 1.5% and 3% of dimethyl sulfoxide can protect the cells well in the gel. This formula can reduce the dimethyl group under the premise of ensuring cell viability. The amount of sulfone used.
  • the experiment is divided into 3 groups:
  • stem cell gel preparation The above components were thoroughly mixed to prepare a stem cell gel preparation, and the stem cell gel preparation was taken as a -1 day count before cryopreservation, and the non-programmed cooling method was frozen at -80 ° C for 0, 1, and 7 days.
  • Three sets of stem cell gel preparations in each group were tested for cell viability and cell yield after resuscitation. It can be seen from the experimental results (Fig. 3) that human albumin can better protect frozen cells and finally select 2% as the concentration of human albumin.
  • the experiment is divided into 5 groups:
  • the above components were thoroughly mixed to prepare a stem cell gel preparation, and the stem cell gel preparation was taken as a -1 day count before cryopreservation, and the non-programmed cooling method was frozen at -80 ° C for 0, 1, and 7 days.
  • Each group of 3 cell gel preparations was tested for cell viability and cell yield after resuscitation. It can be seen from the experimental results (Fig. 4) that the polyphenols can better protect the frozen cells, and finally choose 2% as the concentration of the polyphenol.
  • the experiment is divided into 5 groups:
  • stem cell gel preparation The above components were thoroughly mixed to prepare a stem cell gel preparation, and the stem cell gel preparation was taken as a -1 day count before cryopreservation, and the non-programmed cooling method was frozen at -80 ° C for 0, 1, and 7 days. Three sets of stem cell gel preparations in each group were tested for cell viability and cell yield after resuscitation. It can be seen from the experimental results that dextran can better protect frozen cells, and finally 6% is used as the concentration of dextran (Fig. 5).
  • the experiment is divided into 4 groups:
  • each group was mixed with the above components. Twelve groups of stem cell gel preparations were prepared, and the stem cell gel preparation was taken as a -1 day count before cryopreservation, and frozen at -80 ° C according to a non-programmed cooling method. After 0, 1, and 7 days, three groups of each group were resuscitated and tested for cell viability and cell yield. From the experimental results (Fig. 6), the best gel formulation for maintaining cell viability is 3% sodium alginate, 3% dimethyl sulfoxide, 5% C. Glycol, 2% polyphenol, 6% dextran, 2% human albumin.
  • the gel formulation of the present invention was compared to a control gel formulation in which the control gel formulation was 3% sodium alginate, 10% dimethyl. Sulfoxide, 5% propylene glycol, optimized gel formulation (inventive formulation) is 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 2% polyphenols, 6% dextran and 2% human blood albumin. The immediate recovery and long-term stability of the two formulations were compared. The cell gel preparation was taken as a -1 day count before cryopreservation and frozen at -80 °C according to the non-programmed cooling method.
  • Example 4 Detection of viability of mesenchymal stem cells derived from different tissues in sodium alginate gel
  • the formulation of the invention is 1: 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 2% polyphenols, 6% dextran, 2% human albumin, formula 2: 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 5% polyphenols, 10% dextran, 1% human albumin, Prepared separately into a gel stock solution.
  • the gel stock solution was mixed with bone marrow-derived, umbilical cord-derived, placenta-derived mesenchymal stem cells, and uniformly mixed to prepare a cell gel preparation, which was frozen at -80 ° C according to a non-programmed cooling method. After 0, 1, and 15 days, 3 groups in each group were tested for cell viability and cell yield. From the experimental results (Fig. 8), it can be seen that both sodium alginate gels are good carriers of mesenchymal stem cells derived from various tissues, and are capable of maintaining the activity of mesenchymal stem cells of various sources.
  • the formulation of the invention 1 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 2% polyphenols, 6% dextran, 2% human Serum albumin
  • formula 2 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 5% polyphenols, 10% dextran, 1% human albumin, respectively, formulated into a gel stock solution.
  • the gel stock solution and the mesenchymal stem cells were uniformly mixed, and then frozen at -80 ° C according to a non-programmed cooling method.
  • both cell gel preparations can be stored at -80 °C for 18 months for a long time. Stability.
  • a rat skin lesion model was constructed to produce a 2 cm diameter whole skin defect.
  • Each rat had 2 wounds, including 4 groups, including the blank group and the high dose group (the number of cells was 3 ⁇ 10 6 ).
  • the middle dose group The number of cells (1 ⁇ 10 6 ) and the low dose group (0.2 ⁇ 10 6 ).
  • the wound size was measured every 3-4 days.
  • the extent of skin recovery in each group was compared ( Figure 10). The results showed that the cell gel began to be effective on the third day, and the wound healing rate was faster than that of the blank control group, suggesting that the cell gel preparation can promote wound healing in a dose-dependent manner.

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Abstract

本发明涉及一种保持冻存细胞活性的凝胶制剂和含有干细胞的干细胞凝胶制剂。所述凝胶制剂包括以质量百分比计的下述组分：1％-3％海藻酸钠，1％-5％二甲基亚砜，1％-5％丙二醇，1％-5％益多酚，2％-10％右旋糖酐，1％-5％人血白蛋白，补足水或磷酸盐溶液。该凝胶制剂生物相容性好，操作简便，能够维持细胞的高活性；该干细胞凝胶制剂能直接进行低温保存，不需要传统的液氮冻存，复苏后的细胞凝胶制剂仍能够维持细胞的高活性和细胞的干性，可以应用于制备治疗皮肤损伤或者粘膜损伤的药物。

Description

一种保持冻存细胞活性的干细胞凝胶制剂及其应用 技术领域
本发明涉及生物技术领域，特别涉及一种用于细胞冻存的凝胶制剂；
本发明还涉及含有间充质干细胞和上述凝胶制剂的干细胞凝胶制剂；
本发明还涉及上述干细胞凝胶制剂的应用。
背景技术
组织工程支架材料要作为永久性的替代物，必须是可降解的生物材料，材料本身对组织应该没有免疫原性，而且材料降解的产物对生物组织应该没有毒性或异常反应。胶原虽然对细胞的亲和性好，但常来源于动物组织，病原微生物污染风险大，而且会有免疫原性，且价格较高，力学性能较差，需交联后使用。目前应用于临床的皮肤组织工程产品仍存在一定的免疫排斥反应，治疗效果欠佳。常规处理是在创面上涂外用药消毒、暴露或包扎，但效果并不理想，轻者患者感觉创口疼痛、红肿，重者痂下积脓，肢体活动受限，继而影响正常的工作。
海藻酸钠是从海洋的褐藻中提取的直链阴离子多糖，生物相容性好，具有生物降解吸收性和免疫隔离性能，是目前最常用的生物支架材料。海藻酸钠水溶液为淡黄色半流体胶状液体，可快速均匀的覆盖在创伤表面，减缓表面水分的蒸发，维持细胞活性，对细胞具有保护和支持作用，是细胞外用治疗皮肤组织修复的良好载体，也可以作为肠组织修复和子宫组织修复的细胞支架材料。
间充质细胞因其易于分离、培养、扩增的特点，体外长期培养能够始终保持其干性，具有稳定的遗传背景等独特优势，成为组织工程最好的种子细胞的来源之一。间充质干细胞具有低免疫原性，不会引起机体异常免疫反应；能够释放多种抑炎因子，抑制机体炎症反应，能够释放多种促进组织损伤修复的因子促进组织损伤修复；动员机体自身的干细胞参与组织损伤修复，细胞直接接触缺损或炎性创面可以最大程度的发挥干细胞的修复治疗作用。
保持细胞活性的长期冻存最常用的方法是液氮冻存，能够保持80％以上的细胞活率。但是在细胞制品的运输和临床应用中，液氮不易于通过交通工具运输，细胞液氮冻存在转移运输时受到很大限制。在临床应用时，应用地点需要 配备液氮储存装置和液氮供应，受当地地域和条件限制。
中国专利申请CN 101451124A公开了一种使用甲基纤维素作为细胞基质的人脐带间充质干细胞创面涂抹剂的制备方法，其含有抗生素庆大霉素，不适用于抗生素过敏人群；而且涂抹剂快速配制中含有动物源成分胎牛血清，有可能造成过敏以及具有动物源性安全隐患；快速配制中操作步骤多，过程繁杂，带来微生物污染风险，运输前不能按照药典要求完成2周的无菌检测，制品质量无法保证。
中国专利申请CN 102670654A公开了一种用于创面修复的干细胞制剂，其包含来源于脐带、胎盘或羊膜的间充质干细胞以及高分子稳定剂，所述高分子稳定剂选自海藻酸钠、透明质酸钠、壳聚糖、羟乙基淀粉，还可以含有细胞防冻剂DMSO，保湿剂丙二醇或丙三醇。该制剂虽然具有很好的治疗作用，但其在稳定性和细胞存活率方面还存在一定的不足。
发明内容
本发明的目的在于提供一种用于保持冻存细胞活性的凝胶制剂，其无需液氮冻存能够长期维持细胞高活率，克服了依赖液氮的传统的细胞保存方式，在-80℃条件下能够长期保持细胞高活性。
本发明的另一个目的在于提供一种含有间充质干细胞和所述凝胶制剂的干细胞凝胶制剂。
本发明的再一个目的在于提供所述干细胞凝胶制剂在制备皮肤修复药物中的应用。
根据本发明的一方面，一种用于保持冻存细胞活性的凝胶制剂，包括下述质量百分比的组分：1-3％海藻酸钠，1-5％二甲基亚砜，1-5％丙二醇，1-5％益多酚，2％-10％右旋糖酐，1-5％人血白蛋白，补足水或磷酸盐溶液。其中磷酸盐溶液成分为0.02％氯化钾，0.0047％氯化镁，0.1158％磷酸氢二钠，0.020％磷酸二氢钠，0.8％氯化钠和注射用水。
优选的组成为：3％的海藻酸钠，3％二甲基亚砜，5％丙二醇，2％益多酚，6％右旋糖酐和2％人血白蛋白，补足磷酸盐溶液。其中磷酸盐溶液成分为0.02％氯化钾，0.0047％氯化镁，0.1158％磷酸氢二钠，0.020％磷酸二氢钠，0.8％氯化钠和注射用水。
在现有配方含有海藻酸钠的基础上，本发明的凝胶制剂的组成上增加了益多酚、右旋糖酐和人血白蛋白，益多酚具有非常强的抗氧化活性，抗氧化活性至少是维生素C的100多倍，是维生素E的25倍，能够保护细胞和DNA受损害。右旋糖苷是目前最佳的血浆代用品之一，能够极大的提高细胞活率并降低DMSO的使用量。人血白蛋白的加入使得本发明的凝胶制剂中不含有动物来源成分，提高了制剂的安全性和稳定性。此外，对凝胶制剂各成分配比组成进行了筛选，形成了适于保持冻存细胞活性的凝胶制剂。
本发明的凝胶制剂可用于冻存各种细胞，包括但不限于，各种来源的肿瘤细胞系、动物细胞、干细胞等。
根据本发明的另一方面，提供一种干细胞凝胶制剂，含有间充质干细胞和所述凝胶制剂，优选地，每1ml凝胶制剂中含有0.5～3×106个间充质干细胞。所述间充质干细胞可来源于脐带、胎盘、羊膜组织，通过取上述组织制备，但不限于此，可以是任何来源的间充质干细胞。
本发明所述干细胞凝胶制剂按下述方法配制：将无菌海藻酸钠粉末溶于磷酸盐溶液中，室温溶胀24-48小时后，充分搅拌混匀，再加入其余组分配制成1-3％海藻酸钠凝胶制剂。静置后加入间充质干细胞，混合均匀，制成干细胞凝胶制剂。
为了保证细胞在凝胶中的活力，配制凝胶制剂的海藻酸钠的最适目数为200，粘度600-1000cps。溶液PH值为6.5-7.5。该富含细胞的凝胶制剂可以直接冻存于-80℃～-60℃长期保存，冻存方法为非程序降温。保存期限长达1年半。复苏该富含细胞的凝胶制剂后可以直接使用，细胞活率可以维持在90％以上。如果不立即使用，也可以放置到4℃保存，6小时内细胞活率可以维持在80％以上。
根据本发明的再一方面，提供本发明所述干细胞凝胶制剂在制备治疗皮肤损伤或者粘膜损伤药物中的应用，将含有本发明的凝胶制剂与间充质干细胞的干细胞凝胶制剂制备成适于运输和保存的药物制剂，可用于皮肤修复治疗，所述皮肤修复是指对皮肤损伤或者粘膜损伤的修复。所述皮肤损伤或者粘膜损伤包括但不限于皮肤溃疡，褥疮，糖尿病足，溃疡性结肠炎，克隆氏病，子宫内膜损伤。
本发明的凝胶制剂生物相容性好，能够维持细胞的高活性，不需要液氮冻 存细胞，能直接进行低温保存，便于运输，方便使用。解冻方法简单快速，无需传统方法的洗涤，离心等步骤，减少人为操作对细胞的活率和得率的影响和可能造成污染的风险，解冻后直接使用，复苏后的细胞凝胶制剂仍能够维持细胞的高活性和细胞的干性。
附图说明
图1：不同海藻酸钠材料对细胞活率的影响；
图2：DMSO浓度对细胞活率的影响；
图3：人血白蛋白浓度对细胞活率的影响；
图4：益多酚浓度对细胞活率的影响；
图5：右旋糖酐浓度对细胞活率的影响；
图6：益多酚和右旋糖酐联合使用对细胞活率的影响；
图7：以细胞活率为纵坐标，对比本发明的凝胶配方与对照组的凝胶配方；
图8：不同来源的间充质干细胞在海藻酸钠凝胶中的活率；
图9：长期冻存保存后，本发明的凝胶制剂的细胞活率；
图10：本发明的干细胞凝胶制剂对大鼠皮损的治疗作用。
具体实施方式
以下结合具体实施例，详细描述但不限制本发明。
材料来源：除特别注明外，所有材料均为商业购买。
细胞来源：间充质干细胞来源于但不限于健康人捐献的脐带、胎盘、羊膜组织，经过组织分离、培养扩增、传代获得。
细胞悬液的制备：使用含10％胎牛血清的DMEM培养基(完全培养基)，于37℃、5％CO2的条件下对间充质干细胞进行常规培养，待细胞融合度达到90％左右时，用0.25％的胰蛋白酶进行消化，后用上述完全培养基中止，将所得细胞悬液进行离心，弃上清，再用无血清DMEM培养基对细胞沉淀进行悬浮，调整细胞悬液中间充质干细胞的含量达到0.4×106～2.5×107个/毫升备用。
细胞存活率的计算:细胞存活率(％)＝活细胞总数/(活细胞总数+死细胞总数)×100％
细胞得率的计算：细胞得率(％)＝检测点活细胞总数/初始活细胞总数×100％
【实施例1】：海藻酸钠的筛选
将200目，100目和50目的海藻酸钠粉末分别配制成1％、2％、3％的海藻酸钠溶液，加入3％二甲基亚砜，5％丙二醇，而后加入冻存复苏后的间充质干细胞进行混合，混合均匀后放置在4℃冰箱内，在第0，1，6小时对细胞活率和细胞得率进行比较。由实验结果(图1)可以看出，200目的海藻酸钠是最适合细胞存活的凝胶材料。
【实施例2】：凝胶制剂配方的筛选及优化
为筛选维持细胞活率的最好的凝胶配方，将配方中的组分进行逐一筛选，选出最好的配方：
(1)海藻酸钠凝胶浓度的筛选
将200目的海藻酸钠粉末加入磷酸盐溶液，磷酸盐溶液成分为0.02％氯化钾，0.0047％氯化镁，0.1158％磷酸氢二钠，0.020％磷酸二氢钠，0.8％氯化钠和注射用水。配制成1％，2％，3％，4％的海藻酸钠水溶液，观察凝胶的澄明度，流动性。1％，2％和3％的凝胶的澄明度很好，4％的凝胶中有不溶物。凝胶的流动性随着海藻酸钠浓度的升高而减少，3％的凝胶的流动性最小，附着能力最好。综合澄明度和流动性的结果，最后选择3％作为的海藻酸钠的浓度。
(2)DMSO浓度的筛选
实验分为4组：
Figure PCTCN2017103948-appb-000001
将各组组分充分混匀，制备干细胞凝胶制剂。在冻存前抽取干细胞凝胶制剂作为-1天计数，过夜冻存复苏后第1小时，6小时，24小时取各组3支干细胞凝胶制剂，进行细胞活率和细胞得率检测。由实验结果(图2)可以看出，1.5％和3％的二甲基亚砜在凝胶中均能很好的保护细胞，这个配方中可以在保证细胞活力的前提下减少二甲基亚砜的用量。
(3)人血白蛋白浓度的筛选
实验分为3组：
Figure PCTCN2017103948-appb-000002
各组将以上组分充分混匀，制备干细胞凝胶制剂，在冻存前抽取干细胞凝胶制剂作为-1天计数，非程序降温方法冻存于-80℃后第0，1，7天取各组3支干细胞凝胶制剂，复苏后进行细胞活率和细胞得率检测。由实验结果(图3)可以看出，人血白蛋白能够更好的保护冻存过的细胞，最后选择2％作为人血白蛋白的浓度。
(4)益多酚和右旋糖酐的选择
1)益多酚的浓度筛选
实验分为5组：
Figure PCTCN2017103948-appb-000003
各组将以上组分充分混匀，制备干细胞凝胶制剂，在冻存前抽取干细胞凝胶制剂作为-1天计数，非程序降温方法冻存于-80℃后第0，1，7天取各组3支细胞凝胶制剂，复苏后进行细胞活率和细胞得率检测。由实验结果(图4)可以看出，益多酚能够更好的保护冻存过的细胞，最后选择2％作为益多酚的使用浓度。
2)右旋糖酐的浓度筛选
实验分为5组：
Figure PCTCN2017103948-appb-000004
各组将以上组分充分混匀，制备干细胞凝胶制剂，在冻存前抽取干细胞凝胶制剂作为-1天计数，非程序降温方法冻存于-80℃后第0，1，7天取各组3支干细胞凝胶制剂，复苏后进行细胞活率和细胞得率检测。由实验结果可以看出，右旋糖苷能够更好的保护冻存过的细胞，最后选择6％作为右旋糖酐的使用浓度(图5)。
3)益多酚和右旋糖酐的联合使用
实验分为4组：
Figure PCTCN2017103948-appb-000005
各组将以上组分进行混合。制成干细胞凝胶制剂各组12支，在冻存前抽取干细胞凝胶制剂作为-1天计数，按照非程序降温方法冻存于-80℃。在0，1，7天后，各组各取3支复苏后进行细胞活率和细胞得率检测。由实验结果(图6)可见，维持细胞活率的最好的凝胶配方是3％的海藻酸钠，3％二甲基亚砜，5％丙 二醇，2％益多酚，6％右旋糖酐，2％人血白蛋白。
【实施例3】：优化配方的复苏活率和长期稳定性研究
为研究本发明配方的细胞复苏活率和长期稳定性，用本发明的凝胶配方与对照组凝胶配方做对比，其中，对照组凝胶配方为3％海藻酸钠，10％二甲基亚砜，5％丙二醇，优化的凝胶配方(本发明配方)为3％海藻酸钠，3％二甲基亚砜，5％丙二醇，2％益多酚，6％右旋糖酐和2％人血白蛋白。对两种配方在即时复苏活率和长期稳定性方面进行了比较，在冻存前抽取细胞凝胶制剂作为-1天计数，按照非程序降温方法冻存于-80℃。在0天各组各取3支复苏后，4℃放置6小时后检测细胞活率和细胞得率，后在冻存7，30，90天后各组各取3支复苏后进行细胞活率和细胞得率检测。由实验结果(图7)可见，本发明配方提高了细胞复苏后暂时保存的活率，同时显著的提升了长期保存的能力，使得细胞在非液氮条件下的长期保存成为现实。
【实施例4】：不同组织来源的间充质干细胞在海藻酸钠凝胶中的活率检测
比较两种凝胶配方对不同组织来源的间充质干细胞的活率的影响，本发明配方1：3％海藻酸钠，3％二甲基亚砜，5％丙二醇，2％益多酚，6％右旋糖酐，2％人血白蛋白，配方2：3％的海藻酸钠，3％二甲基亚砜，5％丙二醇，5％益多酚，10％右旋糖酐，1％人血白蛋白，分别配制成凝胶原液。将凝胶原液与骨髓来源的、脐带来源的、胎盘来源的间充质干细胞分别混合，混合均匀后，制成细胞凝胶制剂，按照非程序降温方法冻存于-80℃。在0，1，15天后，各组各取3支进行细胞活率和细胞得率检测。由实验结果(图8)可见，两种海藻酸钠凝胶均是各种组织来源的间充质干细胞的良好载体，能够维持各种不同来源的间充质干细胞的活性。
【实施例5】：长期冻存保存后，凝胶制剂中细胞活率的检测
比较两种凝胶配方对细胞稳定性的影响，本发明配方1：3％的海藻酸钠，3％二甲基亚砜，5％丙二醇，2％益多酚，6％右旋糖酐，2％人血白蛋白，配方2：3％的海藻酸钠，3％二甲基亚砜，5％丙二醇，5％益多酚，10％右旋糖酐，1％人血白蛋白，分别配制成凝胶原液。将凝胶原液和间充质干细胞混合均匀后，按照非程序降温方法冻存于-80℃。在第0，1，3，6，15，18个月，取细胞凝胶制剂3支进行细胞活率和细胞得率检测，并检测其表型，分化能力。由实验结果(图9)得出，这两种细胞凝胶制剂均可以在-80℃长期保存18个月，能够保 持稳定性。
【实施例6】：本发明细胞凝胶制剂对大鼠皮损的治疗作用
构建大鼠皮损模型，制造直径2cm的全皮缺损，每只大鼠有2个创面，共设4个组，包括空白组，高剂量组(细胞数3×106个)，中剂量组细胞数(1×106个)和低剂量组(0.2×106个)。每隔3-4日测量创面大小。比较各组皮肤恢复的程度(图10)。结果表明：细胞凝胶在第3天就开始显效，伤口愈合速度快于空白对照组，提示细胞凝胶制剂能促进伤口愈合，并呈剂量依赖关系。

Claims (10)

  1. 一种保持冻存细胞活性的凝胶制剂，包括以质量百分比计的下述组分：1％-3％海藻酸钠，1％-5％二甲基亚砜，1％-5％丙二醇，1％-5％益多酚，2％-10％右旋糖酐，1％-5％人血白蛋白，补足水或磷酸盐溶液。
  2. 根据权利要求1所述的凝胶制剂，其特征在于所述凝胶制剂包含3％海藻酸钠，3％二甲基亚砜，5％丙二醇，2％益多酚，6％右旋糖酐和2％人血白蛋白，补足水或磷酸盐溶液；
    其中，所述的磷酸盐溶液的成分为0.02％氯化钾，0.0047％氯化镁，0.1158％磷酸氢二钠，0.020％磷酸二氢钠，0.8％氯化钠和注射用水。
  3. 根据权利要求1所述的凝胶制剂，其特征在于所述海藻酸钠的粘度600-1000cps，粒度为200目，所述凝胶制剂PH值为6.5-7.5。
  4. 一种干细胞凝胶制剂，含有间充质干细胞和权利要求1～3任一项所述的凝胶制剂。
  5. 根据权利要求4所述的干细胞凝胶制剂，其特征在于所述制剂中每1ml凝胶制剂中含有0.5×106～3×106个间充质干细胞。
  6. 根据权利要求5所述的干细胞凝胶制剂，其特征在于所述间充质干细胞来源于脐带、胎盘或羊膜间组织。
  7. 根据权利要求4所述的干细胞凝胶制剂，其特征在于该干细胞凝胶制剂经非程序降温冻存于-80℃～-60℃，干细胞保存期限可至18个月，复苏该干细胞凝胶制剂后，细胞活率大于90％，放置到4℃保存，6小时内细胞活率大于80％。
  8. 权利要求4所述的干细胞凝胶制剂的制备方法，包括将海藻酸钠粉末溶于超纯水或磷酸盐溶液中，室温溶胀24-48小时后，充分搅拌混匀，再加入其余组分配制成凝胶制剂，静置后加入间充质干细胞，混合均匀，制成为富含细胞的干细胞凝胶制剂。
  9. 根据权利要求8所述的制备方法，其中所述海藻酸钠的粘度为600-1000cps，粒度为200目，凝胶制剂PH值为6.5-7.5。
  10. 权利要求4所述的干细胞凝胶制剂在制备治疗皮肤损伤或粘膜损伤药物中的应用。
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CN112656815A (zh) * 2020-12-25 2021-04-16 博雅干细胞科技有限公司 治疗宫腔粘连的方法和使用的干细胞制剂
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