CN112471138B - Universal preservation solution for human umbilical cord, amnion and placenta samples and preparation method thereof - Google Patents
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- A—HUMAN NECESSITIES
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- 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
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
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Abstract
Description
技术领域technical field
本发明属于干细胞技术领域,尤其涉及一种人脐带、羊膜和胎盘样本的通用保存液及其制备方法。The invention belongs to the technical field of stem cells, and in particular relates to a universal preservation solution for samples of human umbilical cord, amniotic membrane and placenta and a preparation method thereof.
背景技术Background technique
间充质干细胞是一类具有自我更新和多向分化潜能的细胞,又称多潜能基质细胞,简称MSCs,是属于中胚层的一类多能干细胞,主要存在于结缔组织和器官间质中,包括:骨髓、脐带、脂肪、羊膜、牙髓、胎盘等。在适宜条件下可分化为脂肪、骨、软骨等多种组织细胞。与骨髓来源的MSC相比,脐带、羊膜和胎盘来源的间充质干细胞因其取材方便,无道德伦理争议,可获取的细胞数量多、增殖能力强、免疫调节作用大,分泌细胞生长因子的总量也非常高,便于扩增和传代,同时又没有配型、排异等问题,极其适合用于临床研究和应用,是间充质干细胞的理想来源。Mesenchymal stem cells are a type of cells with self-renewal and multi-directional differentiation potential, also known as pluripotent stromal cells, referred to as MSCs, which are a type of pluripotent stem cells belonging to the mesoderm, mainly present in connective tissues and organ interstitium. Including: bone marrow, umbilical cord, fat, amniotic membrane, dental pulp, placenta, etc. Under suitable conditions, it can differentiate into various tissue cells such as fat, bone and cartilage. Compared with bone marrow-derived MSCs, umbilical cord, amniotic membrane and placenta-derived mesenchymal stem cells are easy to obtain, have no ethical and ethical disputes, can obtain a large number of cells, have a strong proliferation ability, have a strong immune regulation effect, and secrete cell growth factors. The total amount is also very high, which is convenient for expansion and passage, and at the same time, there are no problems such as matching and rejection. It is extremely suitable for clinical research and application, and is an ideal source of mesenchymal stem cells.
但是,间充质干细胞需要通过样本在GMP实验室中制备分离、高效提纯和快速扩增后,才能产出符合标准的细胞。所以,样本的安全保存就成为了间充质干细胞产出的基本要求。而样本从医院产房到GMP实验室,少则需要数小时,多则需要数天,这其中样本会受到诸如时间、温度、营养等因素的影响,导致样本中的细胞数量少,活力低。However, mesenchymal stem cells need to be prepared and isolated in a GMP laboratory, efficiently purified, and rapidly expanded from samples in order to produce cells that meet the standards. Therefore, the safe preservation of samples has become a basic requirement for the production of mesenchymal stem cells. The sample from the hospital delivery room to the GMP laboratory takes at least a few hours to several days. The sample will be affected by factors such as time, temperature, nutrition, etc., resulting in a small number of cells in the sample and low viability.
因此,寻求一种能长时间保存脐带、羊膜和胎盘的通用保存液一直是本领域技术人员亟待解决的问题。Therefore, it has always been an urgent problem for those skilled in the art to seek a universal preservation solution that can preserve the umbilical cord, amniotic membrane and placenta for a long time.
发明内容SUMMARY OF THE INVENTION
有鉴于此,本发明提供了一种人脐带、羊膜和胎盘样本的通用保存液及其制备方法,本通用保存液可用于长时间保存脐带、羊膜和胎盘,获取的间充质干细胞数量多、活率高。In view of this, the present invention provides a universal preservation solution for human umbilical cord, amniotic membrane and placenta samples and a preparation method thereof. The universal preservation solution can be used to preserve the umbilical cord, amniotic membrane and placenta for a long time, and the obtained mesenchymal stem cells are large in number, High survival rate.
本发明的具体技术方案如下:The concrete technical scheme of the present invention is as follows:
一种人脐带、羊膜和胎盘样本的通用保存液,所述通用保存液由DMEM基础培养基、胰岛素、人血白蛋白、维生素C、EGF、青霉素、链霉素和肝素钠组成;A universal preservation solution for samples of human umbilical cord, amniotic membrane and placenta, the universal preservation solution is composed of DMEM basal medium, insulin, human albumin, vitamin C, EGF, penicillin, streptomycin and heparin sodium;
所述通用保存液的pH值为7.5~7.8。The pH value of the universal preservation solution is 7.5-7.8.
优选的,所述胰岛素在所述通用保存液中的浓度为10-20mg/L;Preferably, the concentration of the insulin in the universal preservation solution is 10-20 mg/L;
所述人血白蛋白在所述通用保存液中的浓度为10-15g/L;The concentration of the human albumin in the general preservation solution is 10-15g/L;
所述维生素C在所述通用保存液中的浓度为100-200mg/L;The concentration of the vitamin C in the general preservation solution is 100-200 mg/L;
所述EGF在所述通用保存液中的浓度为10-50μg/L。The concentration of the EGF in the general preservation solution is 10-50 μg/L.
优选的,所述胰岛素在所述通用保存液中的浓度为10mg/L;Preferably, the concentration of the insulin in the universal preservation solution is 10 mg/L;
所述人血白蛋白在所述通用保存液中的浓度为10g/L;The concentration of the human albumin in the universal preservation solution is 10 g/L;
所述维生素C在所述通用保存液中的浓度为100mg/L;The concentration of the vitamin C in the general preservation solution is 100 mg/L;
所述EGF在所述通用保存液中的浓度为10μg/L。The concentration of the EGF in the general preservation solution is 10 μg/L.
优选的,所述青霉素在所述通用保存液中的浓度为50000-100000U/L;Preferably, the concentration of the penicillin in the universal preservation solution is 50,000-100,000 U/L;
所述链霉素在所述通用保存液中的浓度为50-100mg/L;The concentration of the streptomycin in the general preservation solution is 50-100 mg/L;
所述肝素钠在所述通用保存液中的浓度为6250-12500U/L。The concentration of the heparin sodium in the universal preservation solution is 6250-12500 U/L.
优选的,所述青霉素在所述通用保存液中的浓度为100000U/L;Preferably, the concentration of the penicillin in the universal preservation solution is 100,000 U/L;
所述链霉素在所述通用保存液中的浓度为100mg/L;The concentration of the streptomycin in the general preservation solution is 100 mg/L;
所述肝素钠在所述通用保存液中的浓度为6250U/L。The concentration of the heparin sodium in the universal preservation solution is 6250 U/L.
本发明还提供了上述技术方案所述通用保存液的制备方法,包括以下步骤:The present invention also provides the preparation method of the general preservation solution described in the above technical solution, comprising the following steps:
在DMEM基础培养基中加入人血白蛋白和肝素钠,再依次分别加入青霉素、链霉素、胰岛素、维生素C和EGF,然后采用滤膜过滤,得到所述通用保存液。Human serum albumin and heparin sodium were added to the DMEM basal medium, and then penicillin, streptomycin, insulin, vitamin C and EGF were added in sequence, and then filtered through a membrane to obtain the universal preservation solution.
本发明还提供了一种保存样本的方法,包括以下步骤:The present invention also provides a method for preserving the sample, comprising the following steps:
采用上述技术方案所述通用保存液对样本进行保存;The sample is preserved by adopting the universal preservation solution described in the above technical solution;
所述样本为人脐带、羊膜和/或胎盘。The sample is human umbilical cord, amniotic membrane and/or placenta.
优选的,所述样本的目的细胞为间充质干细胞。Preferably, the target cells of the sample are mesenchymal stem cells.
优选的,所述保存的时间为72h以下。Preferably, the storage time is less than 72h.
综上所述,本发明提供了一种通用保存液,所述通用保存液由DMEM基础培养基、胰岛素、人血白蛋白、维生素C、EGF、青霉素、链霉素和肝素钠组成;所述通用保存液的pH值为7.5~7.8。本发明中,DMEM基础培养基为样本的间充质干细胞提供最基础的营养;胰岛素能促进样本间充质干细胞对通用保存液中的糖分进行吸收和利用,维持细胞活性;人血白蛋白为间充质干细胞提供动态平衡,维持细胞渗透压;维生素C能防止长时间的氧化产生的自由基对间充质干细胞造成损伤;EGF能促进间充质干细胞增殖,维持细胞原有形态;青霉素和链霉素可以防止细菌滋生导致的样本污染;肝素钠可以防止血凝结成血块降低新陈代谢;通用保存液的pH值为7.5~7.8,为间充质干细胞提供合适的酸碱环境。实验结果表明,该通用保存液可用于长时间保存脐带、羊膜和胎盘,获取的间充质干细胞数量多、活率高,并且,人血白蛋白、胰岛素、维生素C和EGF在提高保存脐带中间充质干细胞数和间充质干细胞活率具有协同作用。另外,该通用保存液的保质时间可长达半年,可一次性大量配制,控制成本。To sum up, the present invention provides a general preservation solution, which is composed of DMEM basal medium, insulin, human albumin, vitamin C, EGF, penicillin, streptomycin and heparin sodium; the The pH of the general preservation solution is 7.5 to 7.8. In the present invention, the DMEM basal medium provides the most basic nutrition for the mesenchymal stem cells of the sample; insulin can promote the mesenchymal stem cells of the sample to absorb and utilize the sugar in the universal preservation solution to maintain cell activity; human albumin is Mesenchymal stem cells provide dynamic balance and maintain cell osmotic pressure; vitamin C can prevent free radicals generated by long-term oxidation from causing damage to mesenchymal stem cells; EGF can promote the proliferation of mesenchymal stem cells and maintain the original shape of cells; penicillin and Streptomycin can prevent sample contamination caused by bacterial growth; heparin sodium can prevent blood from coagulating into blood clots and reduce metabolism; the pH of the universal preservation solution is 7.5 to 7.8, which provides a suitable acid-base environment for mesenchymal stem cells. The experimental results show that the universal preservation solution can be used to preserve the umbilical cord, amniotic membrane and placenta for a long time, and the obtained mesenchymal stem cells have a large number and high viability. The number of mesenchymal stem cells and the viability of mesenchymal stem cells have a synergistic effect. In addition, the shelf life of the universal preservation solution can be as long as half a year, and can be prepared in large quantities at one time to control costs.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required to be used in the description of the embodiments or the prior art.
图1为本发明实施例2采用不同保存方法保存脐带24h、48h、72h后的间充质干细胞活率图。FIG. 1 is a graph showing the viability of mesenchymal stem cells after umbilical cord was preserved for 24h, 48h and 72h by different preservation methods in Example 2 of the present invention.
具体实施方式Detailed ways
本发明提供了一种人脐带、羊膜和胎盘样本的通用保存液及其制备方法,该通用保存液可用于长时间保存脐带、羊膜和胎盘,获取的间充质干细胞数量多、活率高。The invention provides a universal preservation solution for human umbilical cord, amniotic membrane and placenta samples and a preparation method thereof. The universal preservation solution can be used to preserve the umbilical cord, amniotic membrane and placenta for a long time, and the obtained mesenchymal stem cells have a large number and high viability.
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
实施例1Example 1
本实施例进行通用保存液的制备,包括以下步骤:The present embodiment carries out the preparation of general preservation solution, comprising the following steps:
在950mLDMEM基础培养基中加入50mL20%人血白蛋白(人血白蛋白浓度为10g/50mL)和1mL肝素钠注射液,混匀,再依次分别加入100000U青霉素、100mg链霉素、10mg胰岛素、100mg维生素C和10μgEGF,搅拌溶解,然后采用0.22μm无菌滤膜过滤去除细菌,得到通用保存液,标注生产日期,置于4℃下保存。Add 50mL of 20% human albumin (the concentration of human albumin is 10g/50mL) and 1mL of heparin sodium injection in 950mL DMEM basal medium, mix well, and then add 100000U penicillin, 100mg streptomycin, 10mg insulin, 100mg Vitamin C and 10 μg EGF were dissolved by stirring, and then filtered through a 0.22 μm sterile filter to remove bacteria to obtain a general preservation solution, marked with the production date, and stored at 4°C.
其中,肝素钠注射液的浓度为6250U/mL。Among them, the concentration of heparin sodium injection is 6250U/mL.
对比例1Comparative Example 1
本对比例进行对照通用保存液1的制备,制备方法同实施例1,但对照通用保存液1以等体积的PBS替换50mL20%人血白蛋白。In this comparative example, the control universal preservation solution 1 was prepared, and the preparation method was the same as that of Example 1, but the control universal preservation solution 1 replaced 50 mL of 20% human albumin with an equal volume of PBS.
对比例2Comparative Example 2
本对比例进行对照通用保存液2的制备,制备方法同实施例1,但对照通用保存液2以等质量的PBS替换胰岛素。In this comparative example, the preparation of reference universal preservation solution 2 is carried out, and the preparation method is the same as that of Example 1, but the comparison universal preservation solution 2 is replaced with PBS of the same quality as insulin.
对比例3Comparative Example 3
本对比例进行对照通用保存液3的制备,制备方法同实施例1,但对照通用保存液3以等质量的PBS替换维生素C。In this comparative example, the reference universal preservation solution 3 was prepared, and the preparation method was the same as that of Example 1, but the reference universal preservation solution 3 replaced vitamin C with PBS of the same quality.
对比例4Comparative Example 4
本对比例进行对照通用保存液4的制备,制备方法同实施例1,但对照通用保存液4以等质量的PBS替换EGF。In this comparative example, the preparation of the reference universal preservation solution 4 is carried out, and the preparation method is the same as that of Example 1, but the reference universal preservation solution 4 is replaced with PBS of the same quality as EGF.
实施例2Example 2
在无菌条件下,取剖宫的手术健康、无传染病的新生儿脐带24cm,均匀分成8份,分别保存在实施例1通用保存液、生理盐水、DMEM培养基、无菌容器、对照通用保存液1、对照通用保存液2、对照通用保存液3和对照通用保存液4中,在4℃恒温保存24h、48h、72h后取相同长度的脐带组织使用胶原酶消化法分离脐带间充质干细胞,过滤离心后,使用台盼蓝计数,并计算细胞活率。Under aseptic conditions, take 24cm of umbilical cords of neonates with healthy cesarean section and no infectious diseases, evenly divide them into 8 parts, and store them in the general preservation solution of Example 1, physiological saline, DMEM medium, sterile container, and control universal respectively. Preservation Solution 1, Control Universal Preservation Solution 2, Control Universal Preservation Solution 3, and Control Universal Preservation Solution 4 were stored at 4°C for 24h, 48h, and 72h, and then the same length of umbilical cord tissue was taken to separate umbilical cord mesenchyme by collagenase digestion. Stem cells, filtered and centrifuged, were counted using trypan blue, and cell viability was calculated.
请参阅图1、表1和表2,图1为本发明实施例2采用不同保存方法保存脐带24h、48h、72h后的间充质干细胞活率图,表1为采用不同保存方法保存脐带24h、48h、72h后的间充质干细胞数,表2为采用不同保存方法保存脐带24h、48h、72h后的间充质干细胞活率。结果表明,实施例1通用保存液能在72h内对脐带进行保存,能够在72h内保证分离得到的间充质干细胞有90%以上的活率,能最大限度的保持脐带间充质干细胞的高活性,提高脐带间充质干细胞的提取度,对间充质干细胞后期培养和节约成本有极大的提升。而生理盐水和DMEM基础培养基能在24h内保存脐带,但随着时间的延长,获取的脐带间充质干细胞数量和活率逐步下降;保存在无菌容器中的脐带因为失去液体环境,细胞间大量缺水,导致脐带间充质干细胞快速凋亡,在48h时已经无法分离出活的脐带间充质干细胞。并且,与对照通用保存液1、对照通用保存液2、对照通用保存液3和对照通用保存液4相比,采用实施例1通用保存液保存的脐带中间充质干细胞数和间充质干细胞活率明显更高,表明人血白蛋白、胰岛素、维生素C和EGF在提高保存脐带中间充质干细胞数和间充质干细胞活率具有协同作用。Please refer to Fig. 1, Table 1 and Table 2. Fig. 1 is a graph showing the viability of mesenchymal stem cells after the umbilical cord is preserved for 24h, 48h and 72h by different preservation methods in Example 2 of the present invention. , the number of mesenchymal stem cells after 48h and 72h, and Table 2 shows the viability of mesenchymal stem cells after 24h, 48h and 72h of umbilical cord preservation by different preservation methods. The results show that the universal preservation solution of Example 1 can preserve the umbilical cord within 72 hours, can ensure that the isolated mesenchymal stem cells have a viability of more than 90% within 72 hours, and can maximize the maintenance of the umbilical cord mesenchymal stem cells. It can improve the extraction degree of umbilical cord mesenchymal stem cells, greatly improve the post-cultivation of mesenchymal stem cells and save costs. Physiological saline and DMEM basal medium can preserve the umbilical cord within 24 hours, but with the extension of time, the number and viability of the obtained umbilical cord mesenchymal stem cells gradually decrease; the umbilical cord stored in the sterile container loses the liquid environment, and the cells There was a large amount of water shortage during the period, which led to the rapid apoptosis of umbilical cord mesenchymal stem cells, and the viable umbilical cord mesenchymal stem cells could not be isolated at 48 h. And, compared with the control universal preservation solution 1, the control universal preservation solution 2, the control universal preservation solution 3 and the control universal preservation solution 4, the number of umbilical cord mesenchymal stem cells and the viability of mesenchymal stem cells preserved by the universal preservation solution of Example 1 were compared. The rate was significantly higher, indicating that human serum albumin, insulin, vitamin C and EGF have a synergistic effect in increasing the number of mesenchymal stem cells in the preserved umbilical cord and the viability of mesenchymal stem cells.
表1采用不同保存方法保存脐带24h、48h、72h后的间充质干细胞数Table 1 The number of mesenchymal stem cells after preservation of umbilical cord for 24h, 48h and 72h by different preservation methods
表2采用不同保存方法保存脐带24h、48h、72h后的间充质干细胞活率Table 2 The viability of mesenchymal stem cells after preservation of umbilical cord for 24h, 48h and 72h by different preservation methods
实施例3Example 3
在无菌条件下,取剖宫手术的健康、无传染病的新生儿胎盘一例,将胎盘羊膜剥下,均匀分成8份,分别保存在实施例1通用保存液、生理盐水、DMEM培养基、无菌容器、对照通用保存液1、对照通用保存液2、对照通用保存液3和对照通用保存液4中,在4℃恒温保存24h、48h、72h后取相同质量的羊膜组织使用胶原酶消化法分离羊膜间充质干细胞,过滤离心后,使用台盼蓝计数。Under sterile conditions, a healthy, non-infectious neonatal placenta that underwent cesarean section was taken, the placental amniotic membrane was peeled off, divided into 8 parts, and stored in the general preservation solution of Example 1, normal saline, DMEM medium, Sterile container, control universal preservation solution 1, control universal preservation solution 2, control universal preservation solution 3 and control universal preservation solution 4, store at 4°C for 24h, 48h, and 72h at a constant temperature, take the same quality of amniotic membrane tissue and digest it with collagenase Amniotic membrane-derived mesenchymal stem cells were isolated by filtration, centrifuged, and counted using trypan blue.
结果请参阅表3,结果表明实施例1通用保存液对羊膜有较好的保存效果,在72h仍能分离大量的间充质干细胞,而生理盐水和DMEM基础培养基中的羊膜会随时间的延长细胞数量降低;因羊膜中含有大量的水分,在无菌容器中仍能保存至72h,但间充质干细胞数量会大幅度下降。与对照通用保存液1、对照通用保存液2、对照通用保存液3和对照通用保存液4相比,采用实施例1通用保存液保存的羊膜中间充质干细胞数量明显更大,表明人血白蛋白、胰岛素、维生素C和EGF在提高保存羊膜中间充质干细胞数量具有协同作用。The results are shown in Table 3. The results show that the general preservation solution of Example 1 has a good preservation effect on the amniotic membrane, and a large number of mesenchymal stem cells can still be isolated within 72 hours, while the amniotic membrane in the normal saline and DMEM basal medium will change with time. The number of extended cells is reduced; because the amniotic membrane contains a lot of water, it can still be stored in a sterile container for 72 hours, but the number of mesenchymal stem cells will be greatly reduced. Compared with the control universal preservation solution 1, the control universal preservation solution 2, the control universal preservation solution 3 and the control universal preservation solution 4, the number of amniotic mesenchymal stem cells preserved by the universal preservation solution of Example 1 was significantly larger, indicating that human blood white Protein, insulin, vitamin C and EGF have a synergistic effect in increasing the number of preserved amniotic mesenchymal stem cells.
表3采用不同保存方法保存羊膜24h、48h、72h后的间充质干细胞数Table 3 The number of mesenchymal stem cells after the amniotic membrane was preserved for 24h, 48h and 72h by different preservation methods
实施例4Example 4
在无菌条件下,取剖宫手术的健康、无传染病的新生儿胎盘一例,将胎盘均匀分成8份,分别保存在实施例1通用保存液、生理盐水、DMEM培养基、无菌容器、对照通用保存液1、对照通用保存液2、对照通用保存液3和对照通用保存液4中,在4℃恒温保存24h、48h、72h后取相同质量的胎盘组织使用胶原酶消化法分离胎盘间充质干细胞,过滤离心后,使用台盼蓝计数。Under aseptic conditions, a healthy neonatal placenta without infectious disease was taken by cesarean section, and the placenta was evenly divided into 8 parts, which were respectively stored in the general preservation solution of Example 1, physiological saline, DMEM medium, sterile container, In the control universal preservation solution 1, the control universal preservation solution 2, the control universal preservation solution 3 and the control universal preservation solution 4, the same quality of placental tissue was taken after being stored at a constant temperature of 4 ℃ for 24h, 48h, 72h, and the placental tissue was separated by collagenase digestion method. Mesenchymal stem cells, filtered and centrifuged, were counted using trypan blue.
结果请参阅表4,结果表明实施例1通用保存液对胎盘有较好的保存效果,在72h仍能分离大量的间充质干细胞,而生理盐水、DMEM基础培养基和无菌容器中的胎盘都存在较大的间充质干细胞损失。与对照通用保存液1、对照通用保存液2、对照通用保存液3和对照通用保存液4相比,采用实施例1通用保存液保存的胎盘中间充质干细胞数量明显更大,表明人血白蛋白、胰岛素、维生素C和EGF在提高保存胎盘中间充质干细胞数量具有协同作用。The results are shown in Table 4. The results show that the general preservation solution of Example 1 has a better preservation effect on the placenta, and a large number of mesenchymal stem cells can still be isolated at 72h, while the placenta in the physiological saline, DMEM basal medium and sterile containers can still be isolated. There was a large loss of mesenchymal stem cells. Compared with the control universal preservation solution 1, the control universal preservation solution 2, the control universal preservation solution 3 and the control universal preservation solution 4, the number of placental mesenchymal stem cells preserved by the universal preservation solution of Example 1 was significantly larger, indicating that human blood white Protein, insulin, vitamin C and EGF have synergistic effects in increasing the number of preserved placental mesenchymal stem cells.
表4采用不同保存方法保存胎盘24h、48h、72h后的间充质干细胞数Table 4 The number of mesenchymal stem cells after the placenta was preserved by different preservation methods for 24h, 48h and 72h
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.
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