CN109042624B - Preparation and cryopreservation method and application of human placental subpial large blood vessel tissue - Google Patents
Preparation and cryopreservation method and application of human placental subpial large blood vessel 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
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- 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
Abstract
The invention discloses a method for preparing and cryopreserving human placental sub-chorionic large blood vessel tissues, which comprises the following steps: (1) cleaning the placenta; shearing off slough from placenta edge, and removing amnion; (2) separating chorion of placenta and fetal face from large blood vessel, washing with physiological saline or PBS buffer solution, and washing blood vessel to remove blood clot; then, cutting off the great vessels connected with the chorionic plate one by one; (3) transferring the blood vessel to a freezing tube or a freezing bag, introducing vitrified freezing liquid by a three-step method, transferring to a programmed cooling instrument, cooling to-80 to-90 ℃, and transferring to liquid nitrogen for freezing and storing. The cryopreservation method is beneficial to improving the activity of the cryopreserved tissues and cells, the shape, the function and the structure of the recovered tissues are consistent with those of fresh tissues, 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 sub-chorionic large blood vessel tissues and application thereof, in particular to a method for cryopreserving human placental sub-chorionic large blood vessel tissues and a recovery method thereof.
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. Studies have shown that the better preservation of cells in the form of cryopreserved tissue is that the harmful components of the cryoprotectant do not 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, grind onThe study shows 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 sub-villous membrane large blood vessel tissue, and particularly relates to a cryopreservation method and a recovery method of human placental sub-villous membrane large blood vessel tissue.
The invention is realized by the following technical scheme:
the preparation and cryopreservation method of human placental subpial large blood vessel tissue comprises the following steps:
(1) washing the placenta (to remove dirt and microbial contamination); shearing off the decidua which has fallen off along the edge of the placenta, and removing the amnion;
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 vessel of fetal disc and fetal face with removed amnion, washing with physiological saline or PBS buffer solution, and washing blood vessel to remove blood clot in blood vessel; then, cutting off the large blood vessels connected with the chorion plate one by one (optimally cutting the blood vessels into cylindrical small sections with the length of 2-5 cm);
(3) transferring the blood vessel to a cryopreservation tube or a cryopreservation bag (when transferring, materials such as thin steel wires or plastic wires which can be used in an ultralow temperature environment can pass through the blood vessel to facilitate the transfer), introducing the vitrified cryopreservation liquid by a three-step method, transferring to a programmed cooling instrument, cooling to-80 ℃ to-90 ℃ according to a set cooling program, and transferring to liquid nitrogen for cryopreservation; the cooling procedure is as follows: maintaining at 4 deg.C for 10 min; reducing the temperature to-20 ℃ at the speed of 1 ℃/min; rapidly cooling to-80 to-90 ℃ at the speed of 50-60 ℃/min, keeping the temperature for 24 hours, and rapidly transferring to liquid nitrogen.
The vitrified frozen stock solution consists of a human serum albumin solution with the mass concentration of 20%, dimethyl sulfoxide, propylene glycol, hydroxyethyl urea and trehalose, wherein the mass ratio of each component is as follows: 15-22 wt% of dimethyl sulfoxide, 10-18 wt% of propylene glycol, 10-16 wt% of hydroxyethyl urea, 8-15 wt% of trehalose and the balance of human serum albumin solution. Preferably, the proportion of each component is as follows: 18 wt% of dimethyl sulfoxide, 12 wt% of propylene glycol, 12 wt% of hydroxyethyl urea, 12 wt% of trehalose and the balance of 20% of human serum albumin solution.
The three-step method for introducing the vitrified frozen stock solution comprises the following specific steps:
firstly, introducing trehalose, 50% dimethyl sulfoxide and a part of human serum albumin solution, and balancing for 5min at 4 ℃;
secondly, introducing hydroxyethyl urea, the rest 50 percent of dimethyl sulfoxide and a part of human serum albumin solution, balancing for 2min at 4 ℃,
thirdly, introducing propylene glycol and a part of human serum albumin solution, and immediately putting the propylene glycol and the part of human serum albumin solution into a programmed cooling instrument;
the volume ratio of the liquid introduced for three times is 3:1:1, 20% human serum albumin solution is used for adjusting to proper concentration, and after all the liquid is introduced, the concentration of each component in the vitrified frozen liquid reaches the final concentration requirement.
The large blood vessels under the chorionic membrane are peeled from the placenta, processed into proper size, and then put into a specific container such as a freezing tube or a freezing bag, the specific serial number of the tissue name is coded on the storage container, the sample is frozen to a specific temperature and then put into a specific position of a liquid nitrogen container for long-term storage, and the specific serial number on the container is used for searching the sample during resuscitation.
After the cryopreservation, the resuscitation can be carried out when necessary, and the method comprises the following steps:
taking out the large blood vessel tissue under the placenta chorionic villus from liquid nitrogen, placing the large blood vessel tissue in a gas phase for 10min for balancing, and then quickly placing a freezing bag or a freezing tube 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 macrovascular tissue under the placental chorion by using forceps, putting the macrovascular tissue into a resuscitation solution with a triple standard concentration of 4 ℃ (precooled to 4 ℃) for balancing for 1 minute, taking out the macrovascular tissue, putting the macrovascular tissue into a resuscitation solution with a double standard concentration of 4 ℃ (precooled to 4 ℃) for balancing for 1-3 minutes, taking out the macrovascular tissue, putting the macrovascular tissue 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 chorionic vascular tissue of placenta may be used in separating stem cell related to vascularization.
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 preparation method of the human placental subpial great vessel tissue and the recovery method have the following beneficial effects:
1) the placenta chorion vascular tissue is separated and preserved, and important biological resource is provided 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: HE staining pattern of large vessel tissue under fresh placenta chorion.
FIG. 2 is a HE staining diagram of the recovery of large blood vessel tissue under placental chorionic villi after cryopreservation.
FIG. 3: morphological map of umbilical cord vascular endothelial cells obtained from cryopreserved macrovascular tissue.
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 sub-chorionic large vascular 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 vessel of fetal disc with removed amnion, washing with physiological saline, and flushing blood vessel to remove blood clot; then, cutting off the great vessels connected with the chorion plate one by one (cutting the vessels into cylindrical small sections with the length of 2-5 cm);
(3) transferring the blood vessel into a cryopreservation tube (during transfer, a thin steel wire which can be used in an ultralow temperature environment or a blood vessel is penetrated through the blood vessel to facilitate transfer), introducing vitrified cryopreservation liquid by a three-step method, transferring the vitrified cryopreservation liquid into a programmed cooling instrument, cooling to-80 ℃ according to a set cooling program, and transferring the vitrified cryopreservation liquid to liquid nitrogen for cryopreservation; the cooling procedure is as follows: maintaining at 4 deg.C for 10 min; reducing the temperature to-20 ℃ at the speed of 1 ℃/min; rapidly cooling to-80 deg.C at a rate of 50 deg.C/min, maintaining for 24 hr, and rapidly transferring into liquid nitrogen.
The vitrified frozen stock solution consists of a human serum albumin solution with the mass concentration of 20%, dimethyl sulfoxide, propylene glycol, hydroxyethyl urea and trehalose, wherein the mass ratio of each component is as follows: 18 wt% of dimethyl sulfoxide, 12 wt% of propylene glycol, 12 wt% of hydroxyethyl urea, 12 wt% of trehalose and the balance of 20% of human serum albumin solution.
The three-step method for introducing the vitrified frozen stock solution comprises the following specific steps:
firstly, introducing trehalose, 50% dimethyl sulfoxide and a part of human serum albumin solution, and balancing for 5min at 4 ℃;
secondly, introducing hydroxyethyl urea, the rest 50 percent of dimethyl sulfoxide and a part of human serum albumin solution, balancing for 2min at 4 ℃,
thirdly, introducing propylene glycol and a part of human serum albumin solution, and immediately putting the propylene glycol and the part of human serum albumin solution into a programmed cooling instrument;
the volume ratio of the liquid introduced for three times is 3:1:1, 20% human serum albumin solution is used for adjusting to proper concentration, and after all the liquid is introduced, the concentration of each component in the vitrified frozen liquid reaches the final concentration requirement.
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 large blood vessel tissue under the placenta chorion from liquid nitrogen, standing for 10min to balance, and fast setting the freezing bag in water bath at 37-42 deg.c; after dissolution, quickly transferring the cryopreservation bag into a safety cabinet, opening the cryopreservation bag, lightly taking out large blood vessel tissues under placenta chorions by using forceps, putting the large blood vessel tissues into a resuscitation solution with triple standard concentration at 4 ℃ (precooled to 4 ℃) for balancing for 1 minute, taking out the large blood vessel tissues, putting the large blood vessel tissues into a resuscitation solution with double standard concentration at 4 ℃ (precooled to 4 ℃) for balancing for 1-3 minutes, taking out the large blood vessel tissues, putting the large blood vessel tissues into a 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 recovery liquid consists of trehalose, dextran 40, alanine, glycine and hydroxyethyl starch sodium chloride injection, and the concentration of the recovery liquid is adjusted by taking the hydroxyethyl starch sodium chloride injection as a solvent when the recovery liquid 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;
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;
the concentration of each component in the resuscitation solution with the triple reference concentration is as follows: 15 wt% of trehalose, 18 wt% of dextran 40, 9 wt% of alanine, 9 wt% of glycine and the balance of hydroxyethyl starch sodium chloride injection.
HE staining: sampling fresh placenta chorionic large blood vessel tissue before freezing to perform HE staining; sampling the recovered large blood vessel tissue under the placental chorionic villus, and performing HE staining; the results are shown in fig. 1 and fig. 2, and it can be seen that: the recovered great vessel tissue structure is still maintained, which shows that the cryopreserved recovered great vessel tissue keeps the tissue structure intact. Compared with the HE staining result of the fresh macrovascular tissue, the HE staining of the resuscitation of the macrovascular tissue after cryopreservation is similar to that of the fresh tissue, and no obvious significant difference exists, which indicates that the tissue structure of the resuscitation of the cryopreserved macrovascular tissue is equivalent to that of the fresh tissue.
The frozen large vascular tissue can be used for tissue transplantation after recovery and can also be used for separating umbilical vascular endothelial cells (the separation method is the prior mature technology), as shown in figure 3, the umbilical vascular endothelial cells can be successfully separated after the large vascular tissue frozen by the method of the invention is 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 and cryopreserving human placental subpial large blood vessel tissues 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 and fetal face with removed amnion, washing with physiological saline or PBS buffer solution, and washing blood vessel to remove blood clot in blood vessel; then, cutting off the large blood vessels connected with the chorion plate one by one, and cutting the blood vessels into cylindrical small sections with the length of 2-5 cm;
(3) transferring the blood vessel to a freezing tube or a freezing bag, introducing vitrified freezing liquid by a three-step method, 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: maintaining at 4 deg.C for 10 min; reducing the temperature to-20 ℃ at the speed of 1 ℃/min; rapidly cooling to-80 deg.C at a rate of 50 deg.C/min, maintaining for 24 hr, and rapidly transferring into liquid nitrogen;
the vitrified frozen stock solution consists of a human serum albumin solution with the mass concentration of 20%, dimethyl sulfoxide, propylene glycol, hydroxyethyl urea and trehalose, wherein the mass ratio of each component is as follows: 18 wt% of dimethyl sulfoxide, 12 wt% of propylene glycol, 12 wt% of hydroxyethyl urea, 12 wt% of trehalose and the balance of 20% of human serum albumin solution;
the three-step method for introducing the vitrified frozen stock solution comprises the following specific steps:
firstly, introducing trehalose, 50% dimethyl sulfoxide and a part of human serum albumin solution, and balancing for 10min at 4 ℃;
secondly, introducing hydroxyethyl urea, the rest 50 percent of dimethyl sulfoxide and a part of human serum albumin solution, balancing for 5min at 4 ℃,
thirdly, introducing propylene glycol and a part of human serum albumin solution, and immediately putting the propylene glycol and the part of human serum albumin solution into a programmed cooling instrument;
the volume ratio of the three introduced liquids was 3:1: 1.
2. A method for recovering human placental sub-villous large blood vessel tissue is characterized in that: taking out the placental subpial large blood vessel tissue frozen and preserved by the method for preparing human placental subpial large blood vessel tissue according to claim 1 from liquid nitrogen, placing the frozen and preserved bag or tube in a 37-42 ℃ water bath kettle after the gas phase is left for 10min for balancing; 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 large blood vessel tissue under the placental chorionic villus by using forceps, putting the large blood vessel tissue into a resuscitation solution with a standard concentration three times higher than 4 ℃, balancing for 1 minute, taking out, putting the large blood vessel tissue into a resuscitation solution with a standard concentration two times higher than 4 ℃, balancing for 1-3 minutes, taking out, putting the large blood vessel tissue into a resuscitation solution with a standard concentration one time higher than 4 ℃, balancing for 3 minutes, washing the large blood vessel tissue with a physiological saline or a PBS solution with a temperature of 4 ℃ for 2-5 times, putting the large blood vessel tissue into a physiological saline or a PBS solution with a temperature of 4 ℃, and standing;
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.
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 of preparing human placental sub-chorionic large blood vessel tissue according to claim 1.
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| 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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| CN102899284A (en) * | 2012-10-11 | 2013-01-30 | 天津中医药大学第二附属医院 | Novel in vitro model of human placental barrier |
| 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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