CN108812643B - Preparation and cryopreservation method and application of human placental chorionic villus tissue - Google Patents
Preparation and cryopreservation method and application of human placental chorionic villus tissue Download PDFInfo
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- 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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/34—Sugars
Abstract
The invention discloses a method for preparing human placental chorionic villus tissue for cryopreservation, which comprises the following steps: (1) separating and freezing placenta amnion and decidua; (2) separating and freezing large blood vessel tissues under placenta chorion; (3) separating and freezing placental chorion tissues; (4) and (5) processing and freezing and storing the placenta villus tissue. The invention also provides a method for resuscitation after cryopreservation. The cryopreservation method is beneficial to improving the activity of the cryopreserved tissues and cells, the shape, function and structure of the cryopreserved tissues and the fresh tissues are consistent after recovery, the total survival rate of the cells in the tissues reaches over 90 percent, and the preserved tissues can be used in the fields of stem cell separation, epithelial cell separation and the like, and can also be used in the fields of tissue engineering transplantation and the like.
Description
Technical Field
The invention relates to a method for preparing and cryopreserving human placental chorion tissues and application thereof, in particular to a method for cryopreserving and recovering human placental chorion tissues and a method for recovering human placental chorion tissues.
Background
The placenta is an important organ for material exchange between a fetus and a mother body, and is a tissue-bound organ between the mother and the son, which is formed by the combined growth of an embryonic germ membrane and a mother body endometrium during pregnancy of a human. With the progress of scientific research in recent years, various types of cells including hematopoietic stem cells, mesenchymal stem cells, epithelial cells and the like can be classified from the placenta, thereby changing the state of the placenta as medical waste for a long time.
Modern scientific research suggests that placental amniotic membrane may be used to expand amnion-derived mesenchymal stem cells, as well as in ophthalmic surgery. The placenta chorion can separate out mesenchyme stem cell and hemopoietic stem cell. The villous structure in the placenta is actually capillary vessel tissue from which endothelial stem cells, epithelial stem cells, and the like can be classified. The decidua of placenta is a structure that the placenta is attached to a mother body, and decidua stem cells similar to mesenchymal stem cells are obtained through research. And interstitial tissues rich in mesenchymal stem cells are arranged between the amnion and chorion and between the chorion and decidual. At present, the utilization of the placenta is extensive, one or more stem cells of the placenta are usually separated from only a small part of tissues of the placenta for storage, and the rest part of the placenta is still treated as medical waste, so that waste is generated. Furthermore, with the progress of scientific research, the placenta has functions of hematopoietic organs and the like, can synthesize a large amount of hormones and factors, and contains other nourishing and supporting cells in addition to hematopoietic stem cells.
It should be noted that the methods for extracting stem cells from placenta tissue and preserving the stem cells in the prior art are only performed under the conditions of the prior art means and the cell quality standard, and do not necessarily meet the technical and cell quality standard requirements after the technology advances in the future. Therefore, it is very important to develop a method to freeze and store the tissue of each component of placenta for decades or even long term to preserve its activity, when it is needed in the future, resuscitate the tissue according to the future technical conditions and cell quality standards, and separate again to obtain cells or stem cells more meeting the future requirements, so as to satisfy clinical research and application.
Chinese patent CN201210288706 (grant publication No. CN 102763642B) discloses a freezing protection solution and a method for freezing and preserving human placental amnion and chorion. The size of amnion and chorion preserved by the method is only 1cm2On the left and right sides, the amnion can not be frozen and stored in a large area with complete tissue, the freezing and storing way is simple and single, the survival rate of frozen tissue cells is low, the amnion can only be used for preparing stem cells after amplification, the stored membrane tissue has less living cells, and abundant vascular tissues in the placenta are not stored. However, studies have shown that frozen tissue forms are betterThe activity of preserving cells of (1) is that harmful components in the cryopreservative cannot enter the interstices of the cell-rich membrane structure. Thus, if the volume of cryopreserved tissue is too small, which may expose cells to the toxic components of the cryoprotectant, cell viability may be compromised and the cryoprotectant's cell protective effect may be lost. In addition, researches show that different tissues and even different cells can have good cryopreservation effect only by applying a specific freezing mode, and various tissues of the placenta can be frozen well only by respectively and independently adopting different freezing modes.
Disclosure of Invention
Aiming at the prior art, the invention provides a preparation and cryopreservation method and application of human placental chorion tissues, and particularly relates to a cryopreservation method and a recovery method of human placental chorion tissues.
The invention is realized by the following technical scheme:
a method for preparing human placental chorionic tissue for cryopreservation comprises the following steps:
(1) washing the placenta (to remove dirt and microbial contamination); the decidua which has fallen off is cut off along the edge of the placenta, and the amnion is removed.
The placenta is obtained by collecting the following steps: selecting healthy placenta without infectious diseases and obstetrical complications, and agreeing to a lying-in woman and signing an informed consent form; the collected placenta is transported to a laboratory within 48 hours and subjected to various necessary tests such as detection of infectious diseases such as viruses, detection of bacterial contamination, etc., either normally or by using the placenta collection method described in patent application No. CN 2017106869531 (publication No. 107320332A).
(2) Separating chorion and great blood vessel of placenta with removed amnion, washing with physiological saline or PBS buffer solution, washing blood vessel to remove blood clot in blood vessel, and cutting off great blood vessel connected with chorion plate.
(3) The white tissue which is taken down from the fetal face of the placenta and is left after the amnion and the large blood vessel are removed in the step (2) is the placental chorion tissue (also called chorion plate because of larger volume and thickness), and the chorion plate is longitudinally cut into strip-shaped pieces, wherein the width and the thickness of each strip-shaped piece are 0.5 cm-2 cm, and the length of each strip-shaped piece is 3 cm-15 cm;
putting the cut strip-shaped slices into a freezing bag or a freezing tube, and adding freezing liquid according to the following modes: firstly, adding frozen stock solution 1 at 4 ℃, and balancing for 5 min; secondly, adding the frozen stock solution 2 at the temperature of 0 ℃, and balancing for 15 min; finally, adding the frozen stock solution 3; the volume ratio of the frozen stock solution added for three times is 3:1: 1; then transferring to a programmed cooling instrument, cooling to-80 to-90 ℃ according to a set cooling program, and transferring to liquid nitrogen for freezing and storing;
the cooling procedure is as follows: cooling to 4 ℃, and keeping for 2-5 min; reducing the temperature to 0 ℃ at the speed of 1 ℃/min, and keeping the temperature for 5-10 min; cooling to-10 ℃ within 5-10 min, and keeping for 5-10 min; cooling to-40 ℃ within 40-60 min, then cooling to-80 to-90 ℃ within 1-3 min, and keeping for 5 min.
The frozen stock solution 1 consists of an MEM culture medium and dimethyl sulfoxide, wherein the dimethyl sulfoxide accounts for 5-10 wt%.
The frozen stock solution 2 consists of an MEM culture medium, dimethyl sulfoxide and dextran 40, wherein the dimethyl sulfoxide accounts for 5-10 wt%, and the dextran 40 accounts for 50-70 wt%.
The frozen stock solution 3 (the frozen stock solution in the same step 1) is composed of a human serum albumin solution with the mass concentration of 20%, dimethyl sulfoxide, propylene glycol, hydroxyethyl urea and trehalose, wherein the frozen stock solution comprises the following components in percentage by mass: 5-10 wt% of dimethyl sulfoxide, 5-10 wt% of propylene glycol, 5-10 wt% of hydroxyethyl urea, 5-10 wt% of trehalose and the balance of a human blood albumin solution.
The amnion, decidua, large blood vessel under chorion and chorion tissue are successively stripped from placenta, processed into proper size, and frozen in some specific container, such as freezing tube or freezing bag, with the specific serial number of tissue name being programmed on the container, and the sample is frozen to specific temperature and stored in specific liquid nitrogen container for long term, and the specific serial number in the container is used in searching sample during resuscitation.
After the cryopreservation, the resuscitation can be carried out when necessary, and the method comprises the following steps:
taking out the cryopreserved placental chorionic tissue from liquid nitrogen, placing the cryopreserved placental chorionic tissue in a gas phase for 10min to balance, and then quickly placing a cryopreserved bag or a cryopreserved pipe in a water bath kettle at the temperature of 37-42 ℃, or recovering by adopting a device disclosed in the patent application with the patent application number of 2017107960072 (the publication number of 107365700A); after dissolution, quickly transferring the cryopreservation bag or the cryopreservation tube into a safety cabinet or an ultra-clean bench, opening the cryopreservation bag or the cryopreservation tube, gently taking out the placenta chorion by using forceps, putting the placenta chorion into a resuscitation solution with a triple standard concentration of 4 ℃ (precooled to 4 ℃) for balancing for 1 minute, taking out the placenta chorion, putting the placenta chorion into a resuscitation solution with a double standard concentration of 4 ℃ (precooled to 4 ℃) for balancing for 1-3 minutes, taking out the placenta chorion, putting the placenta chorion into a resuscitation solution with a double standard concentration of 4 ℃ (precooled to 4 ℃) for balancing for 3 minutes, and then putting the placenta chorion into a resuscitation solution with a double standard concentration of 4 ℃ (precooled to 4 ℃) for balancing for 3 minutes; washing with 4 ℃ physiological saline or PBS solution (pre-cooled to 4 ℃) for 2-5 times, putting into 4 ℃ physiological saline or PBS solution (pre-cooled to 4 ℃), and standing for later use;
the recovery solution consists of trehalose, dextran 40, alanine, glycine and hydroxyethyl starch sodium chloride injection, and the concentration of the hydroxyethyl starch sodium chloride injection or MEM culture medium is adjusted by using the hydroxyethyl starch sodium chloride injection or the MEM culture medium as a solvent when the recovery solution is used;
the concentration of each component in the resuscitation solution with one time of reference concentration is as follows: 5 wt% of trehalose, 6 wt% of dextran 40, 3 wt% of alanine, 3 wt% of glycine and the balance of hydroxyethyl starch sodium chloride injection or MEM culture medium;
the concentration of each component in the resuscitation solution with the double reference concentration is as follows: 10 wt% of trehalose, 12 wt% of dextran 40, 6 wt% of alanine, 6 wt% of glycine and the balance of hydroxyethyl starch sodium chloride injection or MEM culture medium;
the concentration of each component in the resuscitation solution with the triple reference concentration is as follows: trehalose accounts for 15 wt%, dextran 40 accounts for 18 wt%, alanine accounts for 9 wt%, glycine accounts for 9 wt%, and the balance is hydroxyethyl starch sodium chloride injection or MEM culture medium.
The recovered placenta chorion can be used for separating placenta chorion mesenchymal stem cells, placenta source hemopoietic stem cells, etc. Or extracting antibody, hormone, etc. from chorion of placenta.
The cryopreservation method of the invention can be used for establishing a placenta-derived tissue or cell resource sample library. The invention lays a solid foundation and basis for perfecting the placenta-derived human genetic resource library.
The freezing storage method and the recovery method for the human placental chorionic tissue have the following beneficial effects:
1) the placenta amnion, decidua, blood vessel under chorion and chorion tissue are separated and preserved separately, and this provides important biological resource for stem cell research from placenta.
2) The placenta membrane tissue is stored completely and in a large area, and the stored tissue can be used in the fields of stem cell separation, epithelial cell separation and the like, and can also be used in the fields of tissue engineering transplantation and the like; in the prior art, various tissues are cut into 1-3 cm3The unified cryopreservation method of the small blocks can only ensure that cells survive on the cell level, can only be used for separating the cells for amplification, and cannot be used in the fields of tissue engineering bulk membrane transplantation and the like.
3) The freezing mode of the invention is beneficial to improving the activity of the frozen tissues and cells. In the prior art, various tissues are cut into 1-3 cm3The unified freezing method of small blocks does not meet the adaptability requirement of each tissue and cell to a specific freezing mode, the tissue activity is poor after freezing, the tissue structure is deformed, and the cell obtaining efficiency is poor.
4) The shape, function and structure of the preserved placenta membrane tissue are consistent with those of a fresh tissue after recovery, and the total survival rate of cells in the tissue reaches more than 90 percent; however, the method for cryopreserving tissue in the prior art generally separates and expands cells to reach a certain value, and actually few surviving cells exist in the recovered tissue, and the separated cells are obtained by proliferating a few surviving cells after the cryopreserved tissue is recovered.
Drawings
FIG. 1: fresh placenta chorion tissue HE staining pattern.
FIG. 2: and (3) recovering the HE staining pattern of the placenta chorion tissues after cryopreservation.
FIG. 3: a mesenchymal stem cell map (upper left) obtained from cryopreserved placental chorionic tissue, an inducible osteoblast map (lower left) and a flow-phenotype map (right).
Detailed Description
The present invention will be further described with reference to the following examples.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
Example 1 cryopreservation of human placental chorionic tissue
Placenta collection: selecting healthy placenta without infectious diseases and obstetrical complications, and agreeing to a lying-in woman and signing an informed consent form; normally collecting placenta, transporting collected placenta to laboratory within 48 hr, and performing various necessary tests such as virus infectious disease test, bacterial contamination test, etc.
The method for freezing preservation comprises the following steps:
(1) washing the placenta (to remove dirt and microbial contamination); the decidua which has fallen off is cut off along the edge of the placenta, and the amnion is removed.
(2) Separating chorion and great blood vessel of placenta with amnion removed, washing with physiological saline, flushing blood vessel to remove blood clots inside the blood vessel, and cutting off the great blood vessel connected to the chorion plate.
(3) The white tissue which is taken down from the fetal face of the placenta and is left after the amnion and the great vessel are removed in the step (2) is the placental chorion tissue (also called chorion plate because of larger volume and thickness), and the chorion plate is longitudinally cut into strip-shaped pieces, wherein the width and the thickness of each strip-shaped piece are 0.5 cm-2 cm, and the length of each strip-shaped piece is 3 cm-15 cm;
putting the cut strip-shaped slices into a freezing storage bag, and adding freezing storage liquid according to the following modes: firstly, adding frozen stock solution 1 at 4 ℃, and balancing for 5 min; secondly, adding the frozen stock solution 2 at the temperature of 0 ℃, and balancing for 15 min; finally, adding the frozen stock solution 3; the volume ratio of the frozen stock solution added for three times is 3:1: 1; then, transferring to a programmed cooling instrument, cooling to-90 ℃ according to a set cooling program, and transferring to liquid nitrogen for freezing and storing;
the cooling procedure is as follows: cooling to 4 deg.C, and maintaining for 2 min; reducing the temperature to 0 ℃ at the speed of 1 ℃/min, and keeping the temperature for 10 min; cooling to-10 deg.C within 10min, and maintaining for 10 min; cooling to-40 deg.C within 45min, cooling to-90 deg.C within 2min, and maintaining for 5 min.
The frozen stock solution 1 consists of an MEM culture medium and dimethyl sulfoxide, wherein the dimethyl sulfoxide accounts for 8 wt%.
The frozen stock solution 2 consists of an MEM culture medium, dimethyl sulfoxide and dextran 40, wherein the dimethyl sulfoxide accounts for 8 wt%, and the dextran 40 accounts for 60 wt%.
The frozen stock solution 3 consists of 20% of human serum albumin solution, dimethyl sulfoxide, propylene glycol, hydroxyethyl urea and trehalose, wherein the frozen stock solution comprises the following components in percentage by mass: 8 wt% of dimethyl sulfoxide, 6 wt% of propylene glycol, 6 wt% of hydroxyethyl urea, 6 wt% of trehalose and the balance of human serum albumin solution.
Example 2 Resuscitation after cryopreservation
After 6 months of cryopreservation according to the method of example 1, resuscitation is performed and mesenchymal stem cells are induced and isolated, as follows: taking out the cryopreserved placental chorion tissues from liquid nitrogen, standing the placenta chorion tissues in a gas phase for 10min to balance, and quickly putting a cryopreservation bag or a cryopreservation tube in a water bath kettle at the temperature of 37-42 ℃; after dissolution, quickly transferring the cryopreservation bag into a safety cabinet, opening the cryopreservation bag, lightly taking out placenta chorion by using forceps, putting the placenta chorion into resuscitation solution with triple standard concentration at 4 ℃ (precooled to 4 ℃) for balancing for 1 minute, taking out, putting the placenta chorion into resuscitation solution with double standard concentration at 4 ℃ (precooled to 4 ℃) for balancing for 1-3 minutes, taking out, putting the placenta chorion into resuscitation solution with double standard concentration at 4 ℃ (precooled to 4 ℃) for balancing for 3 minutes; washing with 4 ℃ physiological saline (pre-cooled to 4 ℃) for 2-5 times, putting into 4 ℃ physiological saline (pre-cooled to 4 ℃) and standing for later use;
the resuscitation solution consists of trehalose, dextran 40, alanine, glycine and hydroxyethyl starch sodium chloride injection;
the concentration of each component in the resuscitation solution with one time of reference concentration is as follows: 5 wt% of trehalose, 6 wt% of dextran 40, 3 wt% of alanine, 3 wt% of glycine and the balance of hydroxyethyl starch sodium chloride injection or MEM buffer solution;
the concentration of each component in the resuscitation solution with the double reference concentration is as follows: 10 wt% of trehalose, 12 wt% of dextran 40, 6 wt% of alanine, 6 wt% of glycine and the balance of hydroxyethyl starch sodium chloride injection or MEM buffer solution;
the concentration of each component in the resuscitation solution with the triple reference concentration is as follows: trehalose accounts for 15 wt%, dextran 40 accounts for 18 wt%, alanine accounts for 9 wt%, glycine accounts for 9 wt%, and the balance is hydroxyethyl starch sodium chloride injection or MEM buffer solution. -
HE staining: fresh placental chorion tissue was sampled for HE staining prior to cryopreservation; sampling the recovered placental chorion tissues, and performing HE staining; the results are shown in fig. 1 and fig. 2, and it can be seen that: the recovered placenta chorion tissue structure is still maintained, which indicates that the placenta chorion tissue after freeze preservation and recovery keeps the tissue structure intact. Compared with the fresh placenta chorion tissue HE staining result, the freeze-stored placenta chorion tissue resuscitation HE staining is similar to that of the fresh tissue, no obvious great difference exists, and the tissue structure of the freeze-stored placenta chorion tissue after resuscitation is equivalent to that of the fresh tissue.
Detecting the survival rate of cells in the resuscitated chorionic tissues: shearing the recovered chorion, adding 0.25% pancreatin digestive juice, digesting at 37 deg.C for 1 hr, adding 2g/L collagenase IV, and digesting at 37 deg.C for two hr. After centrifugation at 1500 rpm, the cells are resuspended by normal saline, sieved by a cell sieve, sampled and stained by trypan blue to detect the cell viability.
The chorion tissue is frozen and stored and recovered by adopting the method disclosed in the embodiment 1 of the Chinese invention patent CN201210288706 (the publication number is CN 102763642B), and the survival rate is detected by adopting the same method, and the result is shown in the table 1, and the cell survival rate in the chorion tissue can reach more than 95 percent and is far higher than that in the prior art through the cell survival rate detection.
TABLE 1 Freeze-stored resuscitative chorionic histocyte viability
The placenta chorion can be separated to obtain the placenta mesenchymal stem cells (the method adopts the prior published technology), the result is shown in figure 3, and the placenta chorion tissues frozen by the method can be successfully separated to obtain the placenta mesenchymal stem cells after being recovered.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each such publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.
Claims (3)
1. A method for preparing human placental chorionic villus tissue is characterized by comprising the following steps: the method comprises the following steps:
(1) cleaning the placenta; shearing off slough from placenta edge, and removing amnion;
(2) separating chorion and great vessel of fetal disc with amnion removed, washing with physiological saline or PBS buffer solution, flushing blood vessel to remove blood clot in blood vessel, and cutting off great vessel connected with chorion plate;
(3) separating and freezing placental chorion tissues: longitudinally cutting the villus membrane plate into strip-shaped pieces, wherein the width and the thickness of each strip-shaped piece are 0.5 cm-2 cm, and the length of each strip-shaped piece is 3 cm-15 cm;
putting the cut strip-shaped slices into a freezing bag or a freezing tube, and adding freezing liquid according to the following modes: firstly, adding frozen stock solution 1 at 4 ℃, and balancing for 5 min; secondly, adding the frozen stock solution 2 at the temperature of 0 ℃, and balancing for 15 min; finally, adding the frozen stock solution 3; the volume ratio of the frozen stock solution added for three times is 3:1: 1; then transferring to a programmed cooling instrument, cooling to-80 to-90 ℃ according to a set cooling program, and transferring to liquid nitrogen for freezing and storing;
the cooling procedure is as follows: cooling to 4 deg.C, and maintaining for 2 min; reducing the temperature to 0 ℃ at the speed of 1 ℃/min, and keeping the temperature for 10 min; cooling to-10 deg.C within 10min, and maintaining for 10 min; cooling to-40 deg.C within 45min, cooling to-90 deg.C within 2min, and maintaining for 5 min;
the frozen stock solution 1 consists of MEM buffer solution and dimethyl sulfoxide, wherein the dimethyl sulfoxide accounts for 8 wt%;
the frozen stock solution 2 consists of an MEM (MEM) buffer solution, dimethyl sulfoxide and dextran 40, wherein the dimethyl sulfoxide accounts for 8 wt%, and the dextran 40 accounts for 60 wt%;
the frozen stock solution 3 consists of 20% of human serum albumin solution, dimethyl sulfoxide, propylene glycol, hydroxyethyl urea and trehalose, wherein the frozen stock solution comprises the following components in percentage by mass: 8 wt% of dimethyl sulfoxide, 6 wt% of propylene glycol, 6 wt% of hydroxyethyl urea, 6 wt% of trehalose and the balance of human serum albumin solution.
2. A method for resuscitating human placental chorionic tissue, comprising: taking the placental chorion tissue frozen and preserved by the method for preparing and cryopreserving human placental chorion tissue according to claim 1 out of liquid nitrogen, standing the liquid nitrogen for 10min to equilibrate, and then rapidly placing a cryopreserving bag or a cryopreserving tube in a water bath kettle at 37-42 ℃; after dissolution, quickly transferring the cryopreservation bag or the cryopreservation tube into a safety cabinet or an ultra-clean bench, opening the cryopreservation bag or the cryopreservation tube, gently taking out placenta chorion by using forceps, putting the placenta chorion into resuscitation liquid with the standard concentration three times of 4 ℃, balancing for 1 minute, taking out, putting the placenta chorion into resuscitation liquid with the standard concentration two times of 4 ℃, balancing for 1-3 minutes, taking out, putting the placenta chorion into resuscitation liquid with the standard concentration one time of 4 ℃, balancing for 3 minutes, washing the placenta chorion with physiological saline or PBS solution with the standard concentration 4 ℃ for 2-5 times, putting the placenta chorion into physiological saline or PBS solution with the standard concentration 4 ℃, and standing;
the recovery solution consists of trehalose, dextran 40, alanine, glycine and hydroxyethyl starch sodium chloride injection, and the concentration of the hydroxyethyl starch sodium chloride injection or MEM culture medium is adjusted by using the hydroxyethyl starch sodium chloride injection or the MEM culture medium as a solvent when the recovery solution is used;
the concentration of each component in the resuscitation solution with one time of reference concentration is as follows: 5 wt% of trehalose, 6 wt% of dextran 40, 3 wt% of alanine, 3 wt% of glycine and the balance of hydroxyethyl starch sodium chloride injection or MEM culture medium;
the concentration of each component in the resuscitation solution with the double reference concentration is as follows: 10 wt% of trehalose, 12 wt% of dextran 40, 6 wt% of alanine, 6 wt% of glycine and the balance of hydroxyethyl starch sodium chloride injection or MEM culture medium;
the concentration of each component in the resuscitation solution with the triple reference concentration is as follows: trehalose accounts for 15 wt%, dextran 40 accounts for 18 wt%, alanine accounts for 9 wt%, glycine accounts for 9 wt%, and the balance is hydroxyethyl starch sodium chloride injection or MEM culture medium.
3. The establishment method of the placenta-derived tissue or cell resource sample library is characterized by comprising the following steps: human placenta is treated and stored by the method for preparing human placental chorionic tissue according to claim 1.
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| CN110892890B (en) * | 2019-12-17 | 2021-09-17 | 银丰低温医学科技有限公司 | Low-temperature preservation method and rewarming method for blood vessels |
| CN112569407B (en) * | 2019-12-25 | 2021-09-10 | 北京光捷扬基健康科技有限公司 | Placenta tissue engineering immunogen-removing skin scaffold and preparation method thereof |
| CN114540291B (en) * | 2022-02-23 | 2024-02-20 | 广西睿健生物科技有限公司 | Umbilical cord mesenchymal stem cell resuscitation protection liquid and resuscitation method |
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| CN102899284B (en) * | 2012-10-11 | 2014-08-06 | 天津中医药大学第二附属医院 | Novel in vitro model of human placental barrier |
| CN103636596B (en) * | 2013-11-29 | 2016-02-24 | 东方康瑞(北京)科技发展有限责任公司 | A kind of biomembrane protection liquid and preparation and application thereof |
| CN104904707A (en) * | 2015-05-29 | 2015-09-16 | 广州赛莱拉干细胞科技股份有限公司 | Composition, application of composition, vitrification cryopreserved agent of placenta and preparation method |
| CN105018417B (en) * | 2015-07-22 | 2018-11-23 | 中国人民解放军第二军医大学 | The load intrinsic stem cell of amnion freezes active amnia particle and its conditioned medium and application |
| CN105123671B (en) * | 2015-07-24 | 2017-10-13 | 广州赛莱拉干细胞科技股份有限公司 | A kind of cells frozen storing liquid, using and immunocyte cryopreservation methods |
| CN107296041B (en) * | 2017-07-02 | 2020-12-25 | 江西瑞济生物工程技术股份有限公司 | Fresh amnion preservation solution, fresh amnion preservation method and application |
| CN108077243A (en) * | 2018-01-24 | 2018-05-29 | 北京臻溪谷医学研究中心（有限合伙） | A kind of freezen protective Human plactnta amnion and chorial protection liquid and preparation method thereof and application method |
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