CN108486057B - Method for extracting hematopoietic stem cells from placenta by using multifunctional placenta tissue processing device - Google Patents

Method for extracting hematopoietic stem cells from placenta by using multifunctional placenta tissue processing device Download PDF

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CN108486057B
CN108486057B CN201810307245.7A CN201810307245A CN108486057B CN 108486057 B CN108486057 B CN 108486057B CN 201810307245 A CN201810307245 A CN 201810307245A CN 108486057 B CN108486057 B CN 108486057B
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placenta
belt
processor
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cylinder
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CN108486057A (en
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张怡
聂艳波
刘艳青
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Tianqing Stem Cell Co ltd
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Tianqing Stem Cell Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Abstract

A method for extracting hematopoietic stem cells in a placenta by using a multifunctional placenta tissue processing device relates to the technical field of biology, in particular to a method for processing placenta tissues. The invention aims to solve the problems that the opening treatment of placenta tissues is easy to pollute when the conventional placenta cells are cleaned and separated, the labor consumption is high in the treatment process, the treatment is incomplete, and the tissue interstitial cells are less to obtain. The placenta tissue processing method comprises the following steps: firstly, cleaning a placenta; secondly, crushing the placenta; and thirdly, collecting the placenta hematopoietic stem cells, namely finishing the tissue treatment of the placenta before the placenta is used for separating the stem cells. The invention is mainly used for carrying out tissue treatment on the placenta.

Description

Method for extracting hematopoietic stem cells from placenta by using multifunctional placenta tissue processing device
Technical Field
The invention relates to the technical field of biology, in particular to a method for processing placenta tissues.
Background
After the fertilized ovum is implanted and the endometrium of the mother body, nutrition and metabolic waste are obtained through the placenta in the process of gradually developing into an individual. Placental tissue is a delicate structure that is an important site for protection and mass energy exchange between the mother and the infant during mammalian growth. The placenta of one term has the diameter of about 160-200mm, the thickness of about 20-25mm and the weight of 650 g, and the structure comprises three parts of amnion, phylliform chorion and decidua basalis. In the chorion, there are a large number of prominent villous leaflets consisting of fetal veins and arteries, so that their specific surface area is greatly increased to about 14 square meters, greatly increasing the area of mass exchange between the fetus and the mother, and thus imparting to the placenta multiple functions, including: 1. natural barrier function. External substances are prevented from directly entering the body of the infant through the filtering and isolating functions of the placenta tissues, such as microorganisms, toxins and the like. 2. And (4) a nutrient exchange function. The nutrition of the maternal sugar, fat, water, minerals, vitamins, antibodies, hormones, amino acids, etc. is transported actively or passively to the capillaries in the placenta to bring the nutrition to the spaces between the villus lobules, and is absorbed by the capillaries in the villus lobules and transported to the fetus. The urea, uric acid, bilirubin, electrolyte, HCG, progesterone, water and the like of the fetus reversely enter maternal blood, so that the excretion of wastes in the fetus is realized. 3. A gas exchange site. Through the placenta, the mother delivers oxygen to the fetus and carbon dioxide is removed from the fetus. 4. Hormone regulation. The hormones in the placenta can play a role in endocrine regulation, and HCG begins to be secreted in the second week of pregnancy and reaches a secretion peak in the 8 th week of pregnancy at the 2 nd week of pregnancy; HCS is expressed starting in the second month of pregnancy and peaking at month 8; the placenta secretes estrogen and progesterone beginning at week 12 of pregnancy, estrogen promotes the development of the myometrium and mammary glands; progesterone can promote the growth of the glandular body in the uterine mucosa, reduce the excitability of the pregnant uterus, reduce early uterine contraction, regulate immunity and the like. 5. The function of hemopoiesis. The transfer of fetal liver hematopoiesis to bone marrow hematopoiesis is completed in the early 34 weeks, and umbilical vein blood contains primitive hematopoietic stem cells and precursor cells which are higher than those of arterial blood in CD34 positive, CD38 negative and have strong colony forming capability, so that the partial hematopoiesis function of the fetus is proved to be from placenta. Histological studies have shown that the placenta contains a large number of stem cells including hematopoietic stem cells, hematopoietic progenitor cells, mesenchymal stem cells, and the like, and has the ability to differentiate into blood cells, osteogenesis, chondrogenesis, fat, and the like, and thus the placenta tissue is also an important tissue material in the field of regenerative medicine.
The placenta is rich in hematopoietic stem cells, 3-5 times of umbilical cord blood, and can be used as main hematopoietic stem cell source in treating blood diseases and immune diseases. The research and application fields of the mesenchymal stem cells obtained after the placenta tissues are cut into pieces and then are separated, cultured and amplified, and the mesenchymal stem cells are used for repairing tissue injuries, such as liver cirrhosis, cartilage injuries, nerve injuries, bone injuries, tendon ligament injuries, skin injuries and the like, and are important stem cell sources for regenerating and repairing organisms.
The stem cells are obtained from the placenta tissue, and various treatments need to be carried out on the placenta tissue, including the steps of washing, disinfecting, cutting the tissue into small pieces, collecting hematopoietic stem cells, culturing the mesenchymal stem cells and the like. The traditional method adopts an open operation method, placenta tissues are placed in a sterile tray, in a safety cabinet, the pretreatment processes of flushing and removing by sterile water, removing after soaking and sterilizing by 75% alcohol, flushing again by sterile water, shearing by manual scissors of the tissues, soaking and separating blood cells by sterile water and the like are sequentially adopted, each process is an open process, and the risk of microbial contamination is very high. Meanwhile, the process needs manpower and high time consumption, and the used instruments also need to be repeatedly cleaned and sterilized, so the cost is high.
Disclosure of Invention
The invention aims to solve the problems that the opening treatment of placenta tissues is easy to pollute when the conventional placenta cells are cleaned and separated, the labor is consumed in the treatment process, the treatment is incomplete and the tissue interstitial cells are less to obtain, and provides a method for extracting hematopoietic stem cells in the placenta by using a multifunctional placenta tissue treatment device.
The method for extracting the hematopoietic stem cells in the placenta by using the multifunctional placenta tissue processing device is specifically completed according to the following steps:
firstly, cleaning a placenta:
the lower end of a processor cylinder body of the placenta processing device is in threaded connection with the upper end of a conical processing liquid receiver of a cleaning liquid receiver, the aseptically collected placenta is placed into the processor cylinder body, the aseptically collected normal saline is injected into the processor cylinder body through a sealing silica gel cap, and the processor cylinder body is sealed; the volume ratio of the sterile normal saline to the processor cylinder is 1 (16-40); the volume ratio of the placenta to the processor barrel is 1 (10-20);
secondly, the connecting pipe and the cleaning fluid guide pipe are respectively arranged on the arc-shaped parts of the first belt and the second belt of the multifunctional electric support in a transverse mode of the processor cylinder; starting an electric controller to enable the processor cylinder to rotate anticlockwise at the speed of 40-60 rpm, meanwhile, the processor cylinder moves longitudinally, the second belt moves downwards when the first belt moves upwards, and washing is carried out for 4-6 min;
taking down the processor cylinder from the multifunctional electric support, vertically placing the processor cylinder in a mode that a sealing silica gel cap is arranged on the processor cylinder, opening a cleaning liquid flow stopping valve, removing the cleaning liquid, closing the cleaning liquid flow stopping valve, injecting sterile normal saline into the processor cylinder through the sealing silica gel cap, and sealing the processor cylinder; the volume ratio of the sterile normal saline to the processor cylinder is 1 (16-40);
fourthly, the connecting pipe and the cleaning fluid guide pipe are respectively arranged on the arc-shaped parts of the first belt and the second belt of the multifunctional electric bracket in a transverse mode of the processor cylinder; starting an electric controller to enable the processor cylinder to rotate anticlockwise at the speed of 40-60 rpm, meanwhile, the processor cylinder moves longitudinally, the second belt moves downwards when the first belt moves upwards, and washing is carried out for 4-6 min;
taking down the processor cylinder from the multifunctional electric bracket, vertically placing the processor cylinder with a closed silica gel cap on the multifunctional electric bracket, opening a cleaning liquid check valve, removing the cleaning liquid, closing the cleaning liquid check valve, injecting 75% alcohol by mass into the processor cylinder through the closed silica gel cap, and sealing the processor cylinder; the volume ratio of the alcohol with the mass fraction of 75% to the volume of the processor cylinder is 1 (15-30);
sixthly, respectively placing the connecting pipe and the cleaning fluid guide pipe on the arc-shaped parts of the first belt and the second belt of the multifunctional electric bracket in a transverse mode of the processor cylinder; starting an electric controller to enable the processor cylinder to rotate anticlockwise at the speed of 40-60 rpm, and meanwhile, the processor cylinder moves longitudinally, and the second belt moves downwards when the first belt moves upwards to soak for 4-6 min;
seventhly, taking down the processor cylinder from the multifunctional electric support, vertically placing the processor cylinder in a mode that a sealing silica gel cap is arranged on the processor cylinder, opening a cleaning liquid flow-stopping valve, removing the sterilized alcohol, injecting sterile normal saline into the processor cylinder through the sealing silica gel cap, and sealing the processor cylinder; the volume ratio of the sterile normal saline to the processor cylinder is 1 (16-40);
placing the connecting pipe and the cleaning liquid guide pipe on arc parts of a first belt and a second belt of the multifunctional electric support respectively in a transverse mode of the processor cylinder; starting an electric controller to enable the processor cylinder to rotate anticlockwise at the speed of 40-60 rpm, meanwhile, the processor cylinder moves longitudinally, the second belt moves downwards when the first belt moves upwards, and washing is carried out for 4-6 min;
ninthly, taking down the processor barrel from the multifunctional electric support, vertically placing the processor barrel in an upper form by a sealing silica gel cap, opening the cleaning liquid flow stopping valve, removing the cleaning liquid, and closing the cleaning liquid flow stopping valve;
secondly, crushing the placenta: the base of the placenta cutting base replaces a conical treatment fluid receiver of a cleaning fluid receiver and is connected with a processor cylinder of a placenta treatment device through threads, sterile normal saline at 4 ℃ is injected into the processor cylinder through a closed silica gel cap, then the base is placed on a multifunctional electric support in a surface contact mode with an electromagnetic stirrer with a platform, the electromagnetic stirrer with the platform is started, a magnetic cutting knife is driven to rotate, the rotating speed is 80-100 rpm, and the treatment is carried out for 5-6 min; the volume ratio of the sterile physiological saline at the temperature of 4 ℃ to the volume of the processor cylinder is 1 (80-100);
thirdly, collecting placenta hematopoietic stem cells:
the method comprises the following steps that firstly, a conical collecting liquid receiver of a hematopoietic cell/tissue collector replaces a base of a placenta shearing base to be in threaded connection with a processor barrel of a placenta processing device, the base is vertically placed in a mode that a closed silica gel cap is arranged on the base, a collecting liquid stop valve is opened, a single cell suspension rich in hematopoietic stem cells is collected, and the collecting liquid stop valve is closed;
injecting placenta tissue enzymolysis liquid into the processor cylinder through a closed silica gel cap, sealing the processor cylinder, and then respectively placing a connecting pipe and a collected liquid guide pipe on arc-shaped parts of a first belt and a second belt of the multifunctional electric bracket in a transverse mode of the processor cylinder; starting an electric controller to enable a processor cylinder to rotate anticlockwise at the speed of 40-60 rpm, meanwhile, the processor cylinder moves longitudinally, a second belt moves downwards when a first belt moves upwards, the processing is carried out for 10-15 min, the processor cylinder is taken down from a multifunctional electric bracket and vertically placed in a mode that a closed silica gel cap is arranged on the multifunctional electric bracket, a collecting liquid stop valve is opened, and the single cell suspension rich in hematopoietic stem cells is collected; the placenta tissue enzymolysis liquid is characterized in that a solvent in the placenta tissue enzymolysis liquid is PBS buffer solution, solutes in the placenta tissue enzymolysis liquid are low-molecular-weight heparin sodium injection, I-type collagenase, II-type collagenase, IV-type collagenase and trypsin-EDTA, the concentration of the low-molecular-weight heparin sodium injection is (2-30) IU/mL, the concentration of the I-type collagenase is (0.05-0.5) mg/mL, the concentration of the II-type collagenase is (0.05-0.5) mg/mL, the concentration of the IV-type collagenase is (0.05-0.5) mg/mL, and the concentration of the trypsin-EDTA is (0.05-0.25) mg/mL; the volume ratio of the placenta tissue enzymolysis liquid to the processor barrel is 1 (20-40);
injecting placenta tissue enzymolysis liquid into the processor cylinder through a closed silica gel cap, sealing the processor cylinder, and then respectively placing a connecting pipe and a collected liquid guide pipe on the arc-shaped parts of a first belt and a second belt of the multifunctional electric bracket in a transverse mode of the processor cylinder; starting an electric controller to enable a processor cylinder to rotate anticlockwise at the speed of 40-60 rpm, meanwhile, the processor cylinder moves longitudinally, a second belt moves downwards when a first belt moves upwards, the processing is carried out for 10-15 min, the processor cylinder is taken down from a multifunctional electric bracket and vertically placed in a mode that a closed silica gel cap is arranged on the multifunctional electric bracket, a collecting liquid stop valve is opened, and the single cell suspension rich in hematopoietic stem cells is collected; the placenta tissue enzymolysis liquid is characterized in that a solvent in the placenta tissue enzymolysis liquid is PBS buffer solution, solutes in the placenta tissue enzymolysis liquid are low-molecular-weight heparin sodium injection, I-type collagenase, II-type collagenase, IV-type collagenase and trypsin-EDTA, the concentration of the low-molecular-weight heparin sodium injection is (2-30) IU/mL, the concentration of the I-type collagenase is (0.05-0.5) mg/mL, the concentration of the II-type collagenase is (0.05-0.5) mg/mL, the concentration of the IV-type collagenase is (0.05-0.5) mg/mL, and the concentration of the trypsin-EDTA is (0.05-0.25) mg/mL; the volume ratio of the placenta tissue enzymolysis liquid to the processor barrel is 1 (20-40);
injecting the PBS buffer solution into the processor cylinder through a sealing silica gel cap, sealing the processor cylinder, and respectively placing the connecting pipe and the collected liquid guide pipe on the arc-shaped parts of the first belt and the second belt of the multifunctional electric bracket in a transverse mode of the processor cylinder; starting an electric controller to enable the processor cylinder to rotate anticlockwise at the speed of 40-60 rpm for processing for 4-6 min; taking down the processor cylinder from the multifunctional electric bracket, vertically placing the processor cylinder in a mode that a closed silica gel cap is arranged on the processor cylinder, opening a collecting liquid stop valve, and collecting the single cell suspension rich in the hematopoietic stem cells; the volume ratio of the PBS buffer solution to the processor cylinder is 1 (10-20);
fifthly, merging the single cell suspension rich in hematopoietic stem cells collected in the third step to the fourth step, namely completing the extraction of stem cells separated by placenta tissue enzymolysis;
the multifunctional placenta tissue processing device comprises a multifunctional electric bracket, a placenta processing device, a cleaning fluid receiver, a placenta shearing base and a hematopoietic cell/tissue collector.
The multifunctional electric support comprises a first belt, an electromagnetic stirrer with a platform, a second belt and an electric controller, wherein the electromagnetic stirrer with the platform is arranged on the electric controller, the first belt and the second belt are arranged on two sides of the electric controller, arc-shaped parts are arranged on the first belt and the second belt, the first belt and the second belt synchronously rotate in an anticlockwise axial direction, and the first belt and the second belt move up and down in a reverse direction;
the placenta treatment device comprises a sealing silica gel cap, a connecting pipe, a processor cover, a processor cylinder and two baffles, wherein the connecting pipe is arranged at the top of the processor cover;
the cleaning solution receiver comprises a conical treatment solution receiver, a 200 micron leakage net, a cleaning solution guide pipe, a cleaning solution latex pipe and a cleaning solution stop valve, wherein the 200 micron leakage net is arranged in the conical treatment solution receiver, the cleaning solution guide pipe is arranged at the bottom end of the conical treatment solution receiver, the tail end of the cleaning solution guide pipe is hermetically connected with the cleaning solution latex pipe, and the cleaning solution stop valve is arranged on the cleaning solution latex pipe;
the placenta cutting base comprises a base and a magnetic shearing knife, the upper end of the base is open, the lower end of the base is closed, and the magnetic shearing knife is arranged at the bottom in the base;
the hematopoietic cell/tissue collector comprises a conical collector receiver, a 70-micron leakage net, a collector draft tube, a collector latex tube and a collector check valve; the 70-micron leakage net is arranged in the conical collecting liquid receiver, the bottom end of the conical collecting liquid receiver is provided with a collecting liquid guide pipe, the tail end of the collecting liquid guide pipe is hermetically connected with a collecting liquid latex pipe, and a collecting liquid stopping valve is arranged on the collecting liquid latex pipe;
when the multifunctional placenta tissue processing device is used for cleaning placenta, the lower end of a processor cylinder of the placenta processing device is in threaded connection with the upper end of a conical processing liquid receiver of a cleaning liquid receiver, and a connecting pipe and a cleaning liquid guide pipe are respectively arranged on the arc-shaped parts of a first belt and a second belt of a multifunctional electric bracket in a transverse mode of the processor cylinder;
when the multifunctional placenta tissue processing device is used for crushing placenta, the lower end of a processor cylinder body of the placenta processing device is in threaded connection with the upper end of a base of a placenta shearing base, and the multifunctional electric bracket is arranged on the multifunctional electric bracket in a mode that the base is in surface contact with an electromagnetic stirrer with a platform;
when the multifunctional placenta tissue processing device is used for collecting the placenta hematopoietic cells/tissues, the lower end of a processor cylinder of the placenta processing device is connected with the upper end of a conical collecting liquid receiver of the hematopoietic cell/tissue collector through threads, and a connecting pipe and a collecting liquid guide pipe are respectively arranged on the arc-shaped parts of a first belt and a second belt of the multifunctional electric bracket in a transverse mode of the processor cylinder.
The invention has the advantages that: the invention relates to a multifunctional placenta tissue processing device, which is a multifunctional processor integrating the functions of cleaning, disinfecting, shearing, separating hematopoietic stem cells and separating placenta tissue blocks. The device can be made in a sterile closed processing mode. The washing, disinfecting, cutting and other processes of the placenta treatment process are traditionally carried out in open trays, and these processes include multiple steps, addition of different reagents, removal of reagents, contamination in open environment due to problems of personnel manipulation or environmental control, and post-hematopoietic contaminationThe sterile isolation and culture of stem cells, mesenchymal stem cells pose a threat. Secondly, the invention relates to a multifunctional placenta tissue processing device, which is an automatic placenta shearing mode. The traditional placenta hematopoietic stem cell separation mode comprises a venous perfusion mode or a manual placenta tissue cutting and crushing mode. The hematopoietic stem cells or precursor cells obtained by the perfusion method are fewer, so most processes adopt the latter manual cutting or crushing method, the labor consumption caused by the manual cutting or crushing method is long, the tissue blocks are not uniform in size and cannot reach the expected size, and meanwhile, the pollution risk of the manual operation and a large amount of manual work such as cleaning of a later-stage operation platform, cleaning of a cutting tool and the like are caused. By adopting an automatic cutting mode, the defects can be completely solved. Thirdly, the multifunctional placenta tissue processing device adopts an electric mixing mode of axial rotation and asynchronous up-and-down turning, is beneficial to repeated contact of cleaning fluid and samples, disinfectant and samples, and collecting fluid and samples, and realizes efficient cleaning and cell recovery functions. Fourthly, the multifunctional placenta tissue processing device of the invention separates hematopoietic stem cells and tissue stem cells simultaneously. The method can obtain the single cell suspension rich in hematopoietic stem cells/precursor cells at one time, and simultaneously separate out the tissue block rich in mesenchymal stem cells which can be directly used for the culture by the adherence method, and has simple and convenient operation. The multifunctional placenta tissue treatment device is simple in structure, low in manufacturing cost and convenient to clean after use, repeated contact between tissues and liquid can be achieved due to the fact that the baffle is arranged in the multifunctional placenta tissue treatment device, the treatment device barrel, the conical treatment liquid receiver and the conical collection liquid receiver are all made of semitransparent epoxy resin materials, the cleaning and disinfection process and the shearing degree of the inner tube disc of the container can be observed conveniently, and high-pressure sterilization can be carried out at the temperature of 121 ℃ before repeated use, so that microbial pollution is avoided. Sixthly, the method can effectively improve the total harvested nucleated cells, and when the volume of the placenta processed once is 500mL, the total harvested nucleated cells once is (1-2) multiplied by 1010The ratio of the seeds reaches more than 13 percent, and the total harvest amount of nucleated cells is (2-5) multiplied by 1010The proportion of each seed reaches more than 28 percent, and the total harvest amount of nucleated cells is more than 5 multiplied by 1010The ratio of the number of the cells reaches more than 57 percent, and the average value of the number of the CD34 positive hematopoietic stem cells reaches 3.2 multiplied by 108The above; the total harvest amount range of the nucleated cells by the conventional method is (2-4) multiplied by 109The number of CD34 positive hematopoietic stem cells is at most 2.8X 107Therefore, the invention adopts soft automatic cutting of placenta tissue, full contact of collected liquid and placenta tissue in a specific belt rotation mode, and solves the problem of less tissue interstitial cells. Seventhly, due to the gentle chopping process and the shortened processing time, the method can effectively improve the survival rate of nucleated cells and the proportion of CD34 positive cells when the volume of the placenta is 500mL after single processing (P)<0.01), compared with the conventional method, the cell viability of the invention averagely rises by 7 percent, which proves that the invention has little damage to cells; the proportion of CD34 positive cells reaches more than 0.54 percent, and is increased by more than 90 percent compared with the proportion of CD34 positive cells by a conventional method; eighthly, the method can effectively reduce the microorganism positive rate, when the volume of the single-time processed placenta is 500mL, the microorganism positive rate of the conventional method reaches more than 28 percent, but the microorganism positive rate of the method is only about 4.5 percent, and the decline trend is obvious; ninth, the invention has significant advantages (P) compared with the conventional method through the comparison of cell clone forming capability (colony forming quantity)<0.05); the invention greatly shortens the time for separating the nucleated cells in the placenta, and the average processing time is 60min and 120 min in the conventional method after the invention is adopted. The time saved mainly comes from the automatic cutting of the placenta, the automatic operation of multiple washing processes (P)<0.01)。
Drawings
Fig. 1 is a schematic view showing the structure of a multifunctional electric stand of the multifunctional placental tissue treatment device according to the present invention, in which fig. 1 shows a first belt, fig. 2 shows an electromagnetic stirrer with a stage attached thereto, fig. 3 shows a second belt, and fig. 4 shows an electric controller;
fig. 2 is a schematic structural view of the placenta treatment device of the multifunctional placenta tissue treatment device of the present invention, wherein 5 denotes a silicone sealing cap, 6 denotes a connecting tube, 7 denotes a processor cover, 8 denotes a processor cylinder, and 9 denotes a baffle plate;
FIG. 3 is a schematic view showing the structure of a washing liquid receiver of the multifunctional placental tissue processing apparatus of the present invention, in which 10 denotes a conical treatment liquid receiver, 11 denotes a 200 μm perforated net, 12 denotes a washing liquid guide pipe, 13 denotes a washing liquid latex pipe, and 14 denotes a washing liquid check valve;
fig. 4 is a schematic view showing a combination structure of a placenta treatment device and a washing liquid receiver when washing placenta, wherein 5 shows a silicone sealing cap, 6 shows a connecting pipe, 7 shows a processor cover, 8 shows a processor cylinder, 9 shows a baffle, 10 shows a conical treatment liquid receiver, 11 shows a 200 micron leaking net, 12 shows a washing liquid guiding pipe, 13 shows a washing liquid emulsion pipe, and 14 shows a washing liquid stopping valve;
fig. 5 is a schematic structural view of a placenta cutting base of the multifunctional placenta tissue treating apparatus of the present invention, in which 15 denotes the base and 16 denotes a magnetic cutting knife;
fig. 6 is a schematic structural view of a hematopoietic cell/tissue collector of the multifunctional placental tissue processing device according to the present invention, in which 17 denotes a conical collector, 18 denotes a 70 μm perforated net, 19 denotes a collector flow guide tube, 20 denotes a collector latex tube, and 21 denotes a collector stop valve.
FIG. 7 is a bar graph of cell viability and expression rate of CD 34-positive cells, in which A is a bar graph of cell viability in example 1, a is a bar graph of cell viability in accordance with a conventional method, B is a bar graph of expression rate of CD 34-positive cells in example 1, and B is a bar graph of expression rate of CD 34-positive cells in accordance with a conventional method;
FIG. 8 is a bar graph of the total number of cell colonies, in which A is a bar graph of the total number of cell colonies in example 1 and a is a bar graph of the total number of cell colonies in the conventional method.
Detailed Description
The first embodiment is as follows: the embodiment is a method for extracting hematopoietic stem cells in placenta by using a multifunctional placenta tissue processing device, which is specifically completed by the following steps:
firstly, cleaning a placenta:
the lower end of a processor cylinder body 8 of the placenta processing device is connected with the upper end of a conical processing liquid receiver 10 of a cleaning liquid receiver through threads, the placenta which is collected in a sterile mode is placed in the processor cylinder body 8, sterile normal saline is injected into the processor cylinder body 8 through a sealing silica gel cap 5, and the processor cylinder body 8 is sealed; the volume ratio of the sterile normal saline to the processor cylinder 8 is 1 (16-40); the volume ratio of the placenta to the processor cylinder 8 is 1 (10-20);
secondly, the connecting pipe 6 and the cleaning fluid guide pipe 12 are respectively arranged on the arc-shaped parts of the first belt 1 and the second belt 3 of the multifunctional electric bracket in a way that the processor cylinder 8 is transversely arranged; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at a speed of 40-60 rpm, meanwhile, the processor cylinder 8 moves longitudinally, and the second belt 3 moves downwards when the first belt 1 moves upwards, and washing is carried out for 4-6 min;
taking down the processor cylinder 8 from the multifunctional electric bracket, vertically placing the processor cylinder in a mode that the sealing silica gel cap 5 is arranged on the multifunctional electric bracket, opening the cleaning liquid check valve 14, removing the cleaning liquid, closing the cleaning liquid check valve 14, injecting sterile normal saline into the processor cylinder 8 through the sealing silica gel cap 5, and sealing the processor cylinder 8; the volume ratio of the sterile normal saline to the processor cylinder 8 is 1 (16-40);
fourthly, the connecting pipe 6 and the cleaning fluid guide pipe 12 are respectively arranged on the arc-shaped parts of the first belt 1 and the second belt 3 of the multifunctional electric bracket in a way that the processor cylinder 8 is transversely arranged; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at a speed of 40-60 rpm, meanwhile, the processor cylinder 8 moves longitudinally, and the second belt 3 moves downwards when the first belt 1 moves upwards, and washing is carried out for 4-6 min;
fifthly, taking the processor cylinder 8 from the multifunctional electric bracket, vertically placing the multifunctional electric bracket with the sealing silica gel cap 5 on, opening the cleaning liquid check valve 14, removing the cleaning liquid, closing the cleaning liquid check valve 14, injecting 75% alcohol into the processor cylinder 8 through the sealing silica gel cap 5, and sealing the processor cylinder 8; the volume ratio of the alcohol with the mass fraction of 75% to the volume of the processor cylinder body 8 is 1 (15-30);
sixthly, respectively placing the connecting pipe 6 and the cleaning solution guide pipe 12 on the arc-shaped parts of the first belt 1 and the second belt 3 of the multifunctional electric bracket in a way that the treater cylinder 8 is transversely placed; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at a speed of 40-60 rpm, meanwhile, the processor cylinder 8 moves longitudinally, and the second belt 3 moves downwards when the first belt 1 moves upwards to soak for 4-6 min;
seventhly, taking down the processor cylinder body 8 from the multifunctional electric support, vertically placing the processor cylinder body in a mode that the sealing silica gel cap 5 is arranged upwards, opening the cleaning liquid stop valve 14, removing the sterilized alcohol, injecting sterile normal saline into the processor cylinder body 8 through the sealing silica gel cap 5, and sealing the processor cylinder body 8; the volume ratio of the sterile normal saline to the processor cylinder 8 is 1 (16-40);
eighthly, respectively placing the connecting pipe 6 and the cleaning liquid guide pipe 12 on arc parts of a first belt 1 and a second belt 3 of the multifunctional electric support in a mode of transversely placing a processor cylinder 8; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at a speed of 40-60 rpm, meanwhile, the processor cylinder 8 moves longitudinally, and the second belt 3 moves downwards when the first belt 1 moves upwards, and washing is carried out for 4-6 min;
ninthly, taking the processor cylinder 8 from the multifunctional electric support, vertically placing the sealing silica gel cap 5 in an upper form, opening the cleaning liquid flow stopping valve 14, removing the cleaning liquid, and closing the cleaning liquid flow stopping valve 14;
secondly, crushing the placenta: replacing a conical treatment fluid receiver 10 of a cleaning fluid receiver with a base 15 of a placenta shearing base to be in threaded connection with a processor cylinder 8 of a placenta treatment device, injecting sterile normal saline at 4 ℃ into the processor cylinder 8 through a closed silica gel cap 5, then placing the base 15 and an electromagnetic stirrer 2 with a platform on a multifunctional electric bracket in a surface contact mode, starting the electromagnetic stirrer 2 with the platform, driving a magnetic shearing knife 16 to rotate at the rotating speed of 80-100 rpm, and treating for 5-6 min; the volume ratio of the sterile physiological saline at the temperature of 4 ℃ to the volume of the processor cylinder body 8 is 1 (80-100);
thirdly, collecting placenta hematopoietic stem cells:
firstly, a conical collecting fluid receiver 17 of a hematopoietic cell/tissue collector replaces a base 15 of a placenta shearing base to be connected with a processor cylinder 8 of a placenta processing device through screw threads, a closed silica gel cap 5 is vertically placed on the conical collecting fluid receiver in an upper mode, a collecting fluid stop valve 21 is opened, a single cell suspension rich in hematopoietic stem cells is collected, and the collecting fluid stop valve 21 is closed;
injecting placenta tissue enzymolysis liquid into a processor cylinder 8 through a closed silica gel cap 5, sealing the processor cylinder 8, and then respectively placing a connecting pipe 6 and a collected liquid guide pipe 19 on arc-shaped parts of a first belt 1 and a second belt 3 of the multifunctional electric bracket in a manner that the processor cylinder 8 is transversely placed; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at a speed of 40-60 rpm, meanwhile, enabling the processor cylinder 8 to move longitudinally, enabling the second belt 3 to move downwards when the first belt 1 moves upwards, processing for 10-15 min, taking down the processor cylinder 8 from the multifunctional electric bracket, vertically placing the processor cylinder 8 in an upper mode through a closed silica gel cap 5, opening the collection liquid flow stopping valve 21, and collecting the single cell suspension rich in the hematopoietic stem cells; the placenta tissue enzymolysis liquid is characterized in that a solvent in the placenta tissue enzymolysis liquid is PBS buffer solution, solutes in the placenta tissue enzymolysis liquid are low-molecular-weight heparin sodium injection, I-type collagenase, II-type collagenase, IV-type collagenase and trypsin-EDTA, the concentration of the low-molecular-weight heparin sodium injection is (2-30) IU/mL, the concentration of the I-type collagenase is (0.05-0.5) mg/mL, the concentration of the II-type collagenase is (0.05-0.5) mg/mL, the concentration of the IV-type collagenase is (0.05-0.5) mg/mL, and the concentration of the trypsin-EDTA is (0.05-0.25) mg/mL; the volume ratio of the placenta tissue enzymolysis liquid to the processor barrel 8 is 1 (20-40);
injecting placenta tissue enzymolysis liquid into a processor cylinder 8 through a closed silica gel cap 5, sealing the processor cylinder 8, and then respectively placing a connecting pipe 6 and a collected liquid guide pipe 19 on the arc-shaped parts of a first belt 1 and a second belt 3 of the multifunctional electric bracket in a manner that the processor cylinder 8 is transversely placed; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at a speed of 40-60 rpm, meanwhile, enabling the processor cylinder 8 to move longitudinally, enabling the second belt 3 to move downwards when the first belt 1 moves upwards, processing for 10-15 min, taking down the processor cylinder 8 from the multifunctional electric bracket, vertically placing the processor cylinder 8 in an upper mode through a closed silica gel cap 5, opening the collection liquid flow stopping valve 21, and collecting the single cell suspension rich in the hematopoietic stem cells; the placenta tissue enzymolysis liquid is characterized in that a solvent in the placenta tissue enzymolysis liquid is PBS buffer solution, solutes in the placenta tissue enzymolysis liquid are low-molecular-weight heparin sodium injection, I-type collagenase, II-type collagenase, IV-type collagenase and trypsin-EDTA, the concentration of the low-molecular-weight heparin sodium injection is (2-30) IU/mL, the concentration of the I-type collagenase is (0.05-0.5) mg/mL, the concentration of the II-type collagenase is (0.05-0.5) mg/mL, the concentration of the IV-type collagenase is (0.05-0.5) mg/mL, and the concentration of the trypsin-EDTA is (0.05-0.25) mg/mL; the volume ratio of the placenta tissue enzymolysis liquid to the processor barrel 8 is 1 (20-40);
injecting the PBS buffer solution into the processor cylinder 8 through the sealing silica gel cap 5, sealing the processor cylinder 8, and respectively placing the connecting pipe 6 and the collected liquid guide pipe 19 on the arc-shaped parts of the first belt 1 and the second belt 3 of the multifunctional electric bracket in a manner that the processor cylinder 8 is transversely placed; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at the speed of 40-60 rpm for 4-6 min; taking down the processor cylinder 8 from the multifunctional electric bracket, vertically placing the processor cylinder in a mode that a closed silica gel cap 5 is arranged on the processor cylinder, opening a collecting liquid stop valve 21, and collecting the single cell suspension rich in the hematopoietic stem cells; the volume ratio of the PBS buffer solution to the volume of the processor cylinder 8 is 1 (10-20);
fifthly, merging the single cell suspension rich in hematopoietic stem cells collected in the third step to the fourth step, namely completing the extraction of stem cells separated by placenta tissue enzymolysis;
the multifunctional placenta tissue processing device comprises a multifunctional electric bracket, a placenta processing device, a cleaning fluid receiver, a placenta shearing base and a hematopoietic cell/tissue collector;
the multifunctional electric support comprises a first belt 1, an electromagnetic stirrer 2 with a platform, a second belt 3 and an electric controller 4, wherein the electromagnetic stirrer 2 with the platform is arranged on the electric controller 4, the first belt 1 and the second belt 3 are arranged on two sides of the electric controller 4, arc-shaped parts are arranged on the first belt 1 and the second belt 3, the first belt 1 and the second belt 3 synchronously rotate in an anticlockwise axial direction, but the first belt 1 and the second belt 3 move up and down in a reverse direction;
the placenta treatment device comprises a sealing silica gel cap 5, a connecting pipe 6, a processor cover 7, a processor barrel 8 and two baffles 9, wherein the connecting pipe 6 is arranged at the top of the processor cover 7, the sealing silica gel cap 5 is arranged at the port of the connecting pipe 6, the lower end of the processor cover 7 is connected with the upper end of the processor barrel 8 through threads, and the two baffles 9 are oppositely arranged on the inner wall of the processor barrel 8;
the cleaning solution receiver comprises a conical treatment solution receiver 10, a 200 micron leakage net 11, a cleaning solution guide pipe 12, a cleaning solution latex pipe 13 and a cleaning solution check valve 14, wherein the 200 micron leakage net 11 is arranged in the conical treatment solution receiver 10, the cleaning solution guide pipe 12 is arranged at the bottom end of the conical treatment solution receiver 10, the tail end of the cleaning solution guide pipe 12 is hermetically connected with the cleaning solution latex pipe 13, and the cleaning solution check valve 14 is arranged on the cleaning solution latex pipe 13;
the placenta cutting base comprises a base 15 and a magnetic shearing knife 16, the upper end of the base 15 is open, the lower end of the base is closed, and the magnetic shearing knife 16 is arranged at the bottom in the base 15;
the hematopoietic cell/tissue collector comprises a conical collecting liquid receiver 17, a 70 micron leakage net 18, a collecting liquid guide pipe 19, a collecting liquid latex pipe 20 and a collecting liquid check valve 21; the 70-micron leakage net 18 is arranged in the conical collection liquid receiver 17, the bottom end of the conical collection liquid receiver 17 is provided with a collection liquid guide pipe 19, the tail end of the collection liquid guide pipe 19 is hermetically connected with a collection liquid latex pipe 20, and the collection liquid latex pipe 20 is provided with a collection liquid check valve 21;
when the multifunctional placenta tissue processing device is used for cleaning placenta, the lower end of a processor cylinder 8 of the placenta processing device is in threaded connection with the upper end of a conical processing liquid receiver 10 of a cleaning liquid receiver, and a connecting pipe 6 and a cleaning liquid guide pipe 12 are respectively arranged on the arc-shaped parts of a first belt 1 and a second belt 3 of a multifunctional electric bracket in a mode that the processor cylinder 8 is transversely placed;
when the multifunctional placenta tissue processing device is used for crushing placenta, the lower end of a processor cylinder 8 of the placenta processing device is in threaded connection with the upper end of a base 15 of a placenta shearing base, and the multifunctional electric bracket is arranged in a mode that the base 15 is in surface contact with an electromagnetic stirrer 2 with a platform;
when the multifunctional placenta tissue processing device is used for collecting the placenta hematopoietic cells/tissues, the lower end of a processor cylinder 8 of the placenta tissue processing device is in threaded connection with the upper end of a conical collecting liquid receiver 17 of the hematopoietic cell/tissue collector, and a connecting pipe 6 and a collecting liquid guide pipe 19 are respectively arranged on the arc-shaped parts of a first belt 1 and a second belt 3 of the multifunctional electric bracket in a mode that the processor cylinder 8 is transversely arranged.
The second embodiment is as follows: the present embodiment differs from the first embodiment in that: the diameter of the arc-shaped part of the first belt 1 is 20cm, and the diameter of the arc-shaped part of the second belt 3 is 20 cm. The rest is the same as the first embodiment.
The third concrete implementation mode: the present embodiment differs from the first or second embodiment in that: the diameter of the platform of the electromagnetic stirrer 2 with the platform is 20 cm. The others are the same as in the first or second embodiment.
The fourth concrete implementation mode: the difference between this embodiment and one of the first to third embodiments is as follows: the lower end of the processor cover 7 is provided with internal threads, and the diameter of the internal threads is 20 cm; the upper and lower of the processor cylinder body 8 are both provided with external threads, the diameter is 20cm, and the height is 25 cm. The other is the same as the first to third embodiments.
The fifth concrete implementation mode: the difference between this embodiment and one of the first to fourth embodiments is: the upper end of the conical treatment liquid receiver 10 is provided with an internal thread with the diameter of 20 cm; the conical liquid collecting receptacle 17 is internally threaded at its upper end and has a diameter of 20 cm. The other is the same as the first to fourth embodiments.
The sixth specific implementation mode: the difference between this embodiment and one of the first to fifth embodiments is as follows: the two baffles 9 are rectangular, and have a length of 10cm and a width of 4 cm. The others are the same as those in the first to fifth embodiments.
The seventh embodiment: the difference between this embodiment and one of the first to sixth embodiments is: the upper end of the base 15 is provided with an internal thread, and the diameter of the internal thread is 20 cm. The other is the same as the first to sixth embodiments.
The specific implementation mode is eight: the difference between this embodiment and one of the first to seventh embodiments is: the magnetic shearing knife 16 is crossed, 8cm in length and 3cm in height. The others are the same as those of the first to seventh embodiments.
The invention is not limited to the above embodiments, and one or a combination of several embodiments may also achieve the object of the invention.
The following tests were carried out to confirm the effects of the present invention
Example 1: the method for extracting the hematopoietic stem cells in the placenta by using the multifunctional placenta tissue processing device is specifically completed according to the following steps:
firstly, cleaning a placenta:
the lower end of a processor cylinder body 8 of the placenta processing device is connected with the upper end of a conical processing liquid receiver 10 of a cleaning liquid receiver through threads, 500mL of aseptically collected placenta is placed into the processor cylinder body 8, 250mL of aseptic normal saline is injected into the processor cylinder body 8 through a closed silica gel cap 5, and the processor cylinder body 8 is sealed;
secondly, the connecting pipe 6 and the cleaning fluid guide pipe 12 are respectively arranged on the arc-shaped parts of the first belt 1 and the second belt 3 of the multifunctional electric bracket in a way that the processor cylinder 8 is transversely arranged; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at a speed of 50rpm, meanwhile, the processor cylinder 8 moves longitudinally, and the second belt 3 moves downwards when the first belt 1 moves upwards, and washing is carried out for 5 min;
taking down the processor cylinder 8 from the multifunctional electric bracket, vertically placing the processor cylinder in a mode that the sealing silica gel cap 5 is arranged on the multifunctional electric bracket, opening the cleaning liquid check valve 14, removing the cleaning liquid, closing the cleaning liquid check valve 14, injecting 250mL of sterile physiological saline into the processor cylinder 8 through the sealing silica gel cap 5, and sealing the processor cylinder 8;
fourthly, the connecting pipe 6 and the cleaning fluid guide pipe 12 are respectively arranged on the arc-shaped parts of the first belt 1 and the second belt 3 of the multifunctional electric bracket in a way that the processor cylinder 8 is transversely arranged; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at a speed of 50rpm, meanwhile, the processor cylinder 8 moves longitudinally, and the second belt 3 moves downwards when the first belt 1 moves upwards, and washing is carried out for 5 min;
fifthly, taking the processor cylinder 8 from the multifunctional electric bracket, vertically placing the multifunctional electric bracket with the sealing silica gel cap 5 on, opening the cleaning liquid check valve 14, removing the cleaning liquid, closing the cleaning liquid check valve 14, injecting 250mL of alcohol with the mass fraction of 75% into the processor cylinder 8 through the sealing silica gel cap 5, and sealing the processor cylinder 8;
sixthly, respectively placing the connecting pipe 6 and the cleaning solution guide pipe 12 on the arc-shaped parts of the first belt 1 and the second belt 3 of the multifunctional electric bracket in a way that the treater cylinder 8 is transversely placed; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at a speed of 50rpm, meanwhile, the processor cylinder 8 moves longitudinally, and the second belt 3 moves downwards when the first belt 1 moves upwards, and is soaked for 5 min;
seventhly, taking down the processor cylinder body 8 from the multifunctional electric support, vertically placing the processor cylinder body in a mode that the sealing silica gel cap 5 is arranged on the multifunctional electric support, opening the cleaning liquid flow-stopping valve 14, removing the sterilized alcohol, injecting 250mL of sterile normal saline into the processor cylinder body 8 through the sealing silica gel cap 5, and sealing the processor cylinder body 8;
eighthly, respectively placing the connecting pipe 6 and the cleaning liquid guide pipe 12 on arc parts of a first belt 1 and a second belt 3 of the multifunctional electric support in a mode of transversely placing a processor cylinder 8; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at a speed of 50rpm, meanwhile, the processor cylinder 8 moves longitudinally, and the second belt 3 moves downwards when the first belt 1 moves upwards, and washing is carried out for 5 min;
ninthly, taking the processor cylinder 8 from the multifunctional electric support, vertically placing the sealing silica gel cap 5 in an upper form, opening the cleaning liquid flow stopping valve 14, removing the cleaning liquid, and closing the cleaning liquid flow stopping valve 14;
secondly, crushing the placenta: replacing a conical treatment fluid receiver 10 of a cleaning fluid receiver with a base 15 of a placenta cutting base to be in threaded connection with a processor cylinder 8 of a placenta treatment device, injecting 100mL of 4-DEG C sterile normal saline into the processor cylinder 8 through a closed silica gel cap 5, then placing the base 15 on a multifunctional electric bracket in a surface contact mode with an electromagnetic stirring machine 2 with a platform, starting the electromagnetic stirring machine 2 with the platform, driving a magnetic shearing knife 16 to rotate at the rotating speed of 90rpm, and treating for 5 min;
thirdly, collecting placenta hematopoietic stem cells:
firstly, a conical collecting fluid receiver 17 of a hematopoietic cell/tissue collector replaces a base 15 of a placenta shearing base to be connected with a processor cylinder 8 of a placenta processing device through screw threads, a closed silica gel cap 5 is vertically placed on the conical collecting fluid receiver in an upper mode, a collecting fluid stop valve 21 is opened, a single cell suspension rich in hematopoietic stem cells is collected, and the collecting fluid stop valve 21 is closed;
injecting 200mL of placenta tissue enzymolysis liquid into a processor cylinder 8 through a closed silica gel cap 5, sealing the processor cylinder 8, and then respectively placing a connecting pipe 6 and a collected liquid guide pipe 19 on arc-shaped parts of a first belt 1 and a second belt 3 of the multifunctional electric bracket in a manner that the processor cylinder 8 is transversely placed; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at the speed of 50rpm, meanwhile, enabling the processor cylinder 8 to move longitudinally, enabling the second belt 3 to move downwards when the first belt 1 moves upwards, processing for 10min, taking down the processor cylinder 8 from the multifunctional electric bracket, vertically placing the processor cylinder 8 in a mode that the closed silica gel cap 5 is arranged upwards, opening the collecting liquid flow stopping valve 21, and collecting the single cell suspension rich in the hematopoietic stem cells; the placenta tissue enzymolysis liquid is characterized in that a solvent in the placenta tissue enzymolysis liquid is PBS buffer solution, solutes are low-molecular-weight heparin sodium injection, I-type collagenase, II-type collagenase, IV-type collagenase and trypsin-EDTA, the concentration of the low-molecular-weight heparin sodium injection is 15IU/mL, the concentration of the I-type collagenase is 0.2mg/mL, the concentration of the II-type collagenase is 0.2mg/mL, the concentration of the IV-type collagenase is 0.2mg/mL, and the concentration of the trypsin-EDTA is 0.1 mg/mL;
injecting 200mL of placenta tissue enzymolysis liquid into a processor cylinder 8 through a closed silica gel cap 5, sealing the processor cylinder 8, and then respectively placing a connecting pipe 6 and a collecting liquid guide pipe 19 on the arc-shaped parts of a first belt 1 and a second belt 3 of the multifunctional electric bracket in a manner that the processor cylinder 8 is transversely placed; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at the speed of 50rpm, meanwhile, enabling the processor cylinder 8 to move longitudinally, enabling the second belt 3 to move downwards when the first belt 1 moves upwards, processing for 10min, taking down the processor cylinder 8 from the multifunctional electric bracket, vertically placing the processor cylinder 8 in a mode that the closed silica gel cap 5 is arranged upwards, opening the collecting liquid flow stopping valve 21, and collecting the single cell suspension rich in the hematopoietic stem cells; the placenta tissue enzymolysis liquid is characterized in that a solvent in the placenta tissue enzymolysis liquid is PBS buffer solution, solutes are low-molecular-weight heparin sodium injection, I-type collagenase, II-type collagenase, IV-type collagenase and trypsin-EDTA, the concentration of the low-molecular-weight heparin sodium injection is 15IU/mL, the concentration of the I-type collagenase is 0.2mg/mL, the concentration of the II-type collagenase is 0.2mg/mL, the concentration of the IV-type collagenase is 0.2mg/mL, and the concentration of the trypsin-EDTA is 0.1 mg/mL;
injecting 200mL of PBS buffer solution into the processor cylinder 8 through the sealing silica gel cap 5, sealing the processor cylinder 8, and respectively placing the connecting pipe 6 and the collected liquid guide pipe 19 on the arc-shaped parts of the first belt 1 and the second belt 3 of the multifunctional electric bracket in a manner that the processor cylinder 8 is transversely placed; starting the electric controller 4 to enable the processor cylinder 8 to rotate anticlockwise at the speed of 50rpm for processing for 5 min; taking down the processor cylinder 8 from the multifunctional electric bracket, vertically placing the processor cylinder in a mode that a closed silica gel cap 5 is arranged on the processor cylinder, opening a collecting liquid stop valve 21, and collecting the single cell suspension rich in the hematopoietic stem cells;
fifthly, merging the single cell suspension rich in hematopoietic stem cells collected in the third step to the fourth step, namely completing the extraction of stem cells separated by placenta tissue enzymolysis;
the multifunctional placenta tissue processing device comprises a multifunctional electric bracket, a placenta processing device, a cleaning fluid receiver, a placenta shearing base and a hematopoietic cell/tissue collector;
the multifunctional electric support comprises a first belt 1, an electromagnetic stirrer 2 with a platform, a second belt 3 and an electric controller 4, wherein the electromagnetic stirrer 2 with the platform is arranged on the electric controller 4, the first belt 1 and the second belt 3 are arranged on two sides of the electric controller 4, arc-shaped parts are arranged on the first belt 1 and the second belt 3, the first belt 1 and the second belt 3 synchronously rotate in an anticlockwise axial direction, but the first belt 1 and the second belt 3 move up and down in a reverse direction;
the placenta treatment device comprises a sealing silica gel cap 5, a connecting pipe 6, a processor cover 7, a processor barrel 8 and two baffles 9, wherein the connecting pipe 6 is arranged at the top of the processor cover 7, the sealing silica gel cap 5 is arranged at the port of the connecting pipe 6, the lower end of the processor cover 7 is connected with the upper end of the processor barrel 8 through threads, and the two baffles 9 are oppositely arranged on the inner wall of the processor barrel 8;
the cleaning solution receiver comprises a conical treatment solution receiver 10, a 200 micron leakage net 11, a cleaning solution guide pipe 12, a cleaning solution latex pipe 13 and a cleaning solution check valve 14, wherein the 200 micron leakage net 11 is arranged in the conical treatment solution receiver 10, the cleaning solution guide pipe 12 is arranged at the bottom end of the conical treatment solution receiver 10, the tail end of the cleaning solution guide pipe 12 is hermetically connected with the cleaning solution latex pipe 13, and the cleaning solution check valve 14 is arranged on the cleaning solution latex pipe 13;
the placenta cutting base comprises a base 15 and a magnetic shearing knife 16, the upper end of the base 15 is open, the lower end of the base is closed, and the magnetic shearing knife 16 is arranged at the bottom in the base 15;
the hematopoietic cell/tissue collector comprises a conical collecting liquid receiver 17, a 70 micron leakage net 18, a collecting liquid guide pipe 19, a collecting liquid latex pipe 20 and a collecting liquid check valve 21; the 70-micron leakage net 18 is arranged in the conical collection liquid receiver 17, the bottom end of the conical collection liquid receiver 17 is provided with a collection liquid guide pipe 19, the tail end of the collection liquid guide pipe 19 is hermetically connected with a collection liquid latex pipe 20, and the collection liquid latex pipe 20 is provided with a collection liquid check valve 21;
when the multifunctional placenta tissue processing device is used for cleaning placenta, the lower end of a processor cylinder 8 of the placenta processing device is in threaded connection with the upper end of a conical processing liquid receiver 10 of a cleaning liquid receiver, and a connecting pipe 6 and a cleaning liquid guide pipe 12 are respectively arranged on the arc-shaped parts of a first belt 1 and a second belt 3 of a multifunctional electric bracket in a mode that the processor cylinder 8 is transversely placed;
when the multifunctional placenta tissue processing device is used for crushing placenta, the lower end of a processor cylinder 8 of the placenta processing device is in threaded connection with the upper end of a base 15 of a placenta shearing base, and the multifunctional electric bracket is arranged in a mode that the base 15 is in surface contact with an electromagnetic stirrer 2 with a platform;
when the multifunctional placenta tissue processing device is used for collecting the placenta hematopoietic cells/tissues, the lower end of a processor cylinder 8 of the placenta tissue processing device is in threaded connection with the upper end of a conical collecting liquid receiver 17 of the hematopoietic cell/tissue collector, and a connecting pipe 6 and a collecting liquid guide pipe 19 are respectively arranged on the arc-shaped parts of a first belt 1 and a second belt 3 of the multifunctional electric bracket in a mode that the processor cylinder 8 is transversely arranged.
In this embodiment, the diameter of the arc part of the first belt 1 is 20cm, and the diameter of the arc part of the second belt 3 is 20 cm.
The diameter of the platform of the electromagnetic stirrer 2 with the platform in this embodiment is 20 cm.
In the embodiment, the lower end of the processor cover 7 is provided with internal threads, and the diameter of the internal threads is 20 cm; the upper and lower of the processor cylinder body 8 are both provided with external threads, the diameter is 20cm, and the height is 25 cm.
The upper end of the conical treatment liquid receiver 10 of the present embodiment is provided with an internal thread, and the diameter is 20 cm; the conical liquid collecting receptacle 17 is internally threaded at its upper end and has a diameter of 20 cm.
In this embodiment, the two baffles 9 are rectangular, and have a length of 10cm and a width of 4 cm.
In this embodiment, the upper end of the base 15 is provided with an internal thread, and the diameter of the internal thread is 20 cm.
In this embodiment, the magnetic shearing knife 16 is crossed, and has a length of 8cm and a height of 3 cm.
The operation of example 1 is repeated to obtain 100 parts of single cell suspension rich in hematopoietic stem cells; adopting the conventional method, taking 500mL of aseptically collected placenta as an initial raw material, and obtaining 100 parts of single cell suspension rich in hematopoietic stem cells; the total amount of harvested nucleated cells was counted, and in example 1, the total amount of harvested nucleated cells was (1-2). times.1010The ratio of each of the cells was 13.5%, and the total amount of nucleated cells harvested (2-5). times.1010The ratio of each of the cells is 28.6%, and the total amount of harvested nucleated cells is > 5X 1010The ratio of the number of the cells is 57.9 percent, and the average value of the number of the CD34 positive hematopoietic stem cells reaches 3.2 multiplied by 108More than one; the conventional method, such as "novel method for extracting hematopoietic stem cells from placenta tissue and using the hematopoietic stem cells in hematopoietic stem cell bank" has a range of total nucleated cell harvest of (2-4) × 109The number of CD34 positive hematopoietic stem cells is at most 2.8X 107And (4) respectively.
For the statistics of the cell viability and the expression rate of CD34 positive cells of 100 single cell suspensions rich in hematopoietic stem cells obtained in example 1 and 100 single cell suspensions rich in hematopoietic stem cells obtained by the conventional method, as shown in FIG. 7, FIG. 7 is a bar graph of the cell viability and the expression rate of CD34 positive cells, wherein A is a bar graph of the cell viability of example 1, a is a bar graph of the cell viability of the conventional method, B is a bar graph of the expression rate of CD34 positive cells of example 1, and B is a bar graph of the expression rate of CD34 positive cells of the conventional method; as can be seen from FIG. 7, due to the mild mincing process and the shortened treatment time, the method of the present invention can effectively improve the survival rate of nucleated cells and the ratio of CD34 positive cells, and compared with the conventional method, the average increase of the cell survival rate is 7%, which proves that the method of the present invention has less damage to the cells than the conventional method; the proportion of CD34 positive cells reaches more than 0.54 percent, and is increased by more than 90 percent compared with the proportion of CD34 positive cells by a conventional method.
The bacterial contamination rate (microorganism positive rate) statistics of 100 parts of the single cell suspension rich in the hematopoietic stem cells obtained in example 1 and 100 parts of the single cell suspension rich in the hematopoietic stem cells obtained by the conventional method is carried out, the bacterial contamination rate of example 1 is 4.88%, and the bacterial contamination rate of the conventional method reaches 28.13%, so that the microorganism positive rate can be effectively reduced by comparing.
Taking 100 microliter of single cell suspension rich in hematopoietic stem cells, performing erythrocyte lysis, washing, counting, adding into human whole-line methyl cellulose basal medium at 37 ℃ and 5% CO2Culturing for 14-16 days in a saturated humidity environment, observing by using an inverted microscope, recording results, and calculating the number of colony cells which can be differentiated from every ten thousand cells. As shown in FIG. 8, FIG. 8 is a bar graph of the total number of cell colonies, wherein A is a bar graph of the total number of cell colonies in example 1 and a is a bar graph of the total number of cell colonies in the conventional method, and it was confirmed from FIG. 8 that the nucleated cells obtained by the conventional method of the present invention have a higher content of stem/progenitor cells and well preserved functions, and the stem/progenitor cells have a strong ability to differentiate into blood cells.

Claims (8)

1. The method for extracting the hematopoietic stem cells in the placenta by using the multifunctional placenta tissue processing device is characterized by comprising the following steps of:
firstly, cleaning a placenta:
the lower end of a processor cylinder body (8) of the placenta processing device is connected with the upper end of a conical processing liquid receiver (10) of a cleaning liquid receiver through threads, the placenta which is collected in a sterile mode is placed into the processor cylinder body (8), sterile normal saline is injected into the processor cylinder body (8) through a sealing silica gel cap (5), and the processor cylinder body (8) is sealed; the volume ratio of the sterile normal saline to the processor cylinder (8) is 1 (16-40); the volume ratio of the placenta to the processor barrel (8) is 1 (10-20);
secondly, the connecting pipe (6) and the cleaning fluid guide pipe (12) are respectively arranged on the arc-shaped parts of the first belt (1) and the second belt (3) of the multifunctional electric support in a transverse mode of the processor cylinder (8); starting the electric controller (4), enabling the processor cylinder (8) to rotate anticlockwise at the speed of 40-60 rpm, enabling the processor cylinder (8) to move longitudinally, enabling the second belt (3) to move downwards when the first belt (1) moves upwards, and washing for 4-6 min;
taking down the processor cylinder (8) from the multifunctional electric support, vertically placing the processor cylinder in a mode that the sealing silica gel cap (5) is arranged on the multifunctional electric support, opening the cleaning liquid check valve (14), discharging the cleaning liquid, closing the cleaning liquid check valve (14), injecting sterile normal saline into the processor cylinder (8) through the sealing silica gel cap (5), and sealing the processor cylinder (8); the volume ratio of the sterile normal saline to the processor cylinder (8) is 1 (16-40);
fourthly, the connecting pipe (6) and the cleaning fluid guide pipe (12) are respectively arranged on the arc-shaped parts of the first belt (1) and the second belt (3) of the multifunctional electric bracket in a transverse mode of the processor cylinder (8); starting the electric controller (4), enabling the processor cylinder (8) to rotate anticlockwise at the speed of 40-60 rpm, enabling the processor cylinder (8) to move longitudinally, enabling the second belt (3) to move downwards when the first belt (1) moves upwards, and washing for 4-6 min;
taking down the processor cylinder (8) from the multifunctional electric support, vertically placing the processor cylinder with a closed silica gel cap (5) on the multifunctional electric support, opening a cleaning liquid check valve (14), discharging the cleaning liquid, closing the cleaning liquid check valve (14), injecting 75% alcohol by mass into the processor cylinder (8) through the closed silica gel cap (5), and sealing the processor cylinder (8); the volume ratio of the alcohol with the mass fraction of 75% to the volume of the processor cylinder (8) is 1 (15-30);
sixthly, the connecting pipe (6) and the cleaning liquid guide pipe (12) are respectively arranged on the arc-shaped parts of the first belt (1) and the second belt (3) of the multifunctional electric bracket in a transverse mode of the processor cylinder (8); starting the electric controller (4), enabling the processor cylinder (8) to rotate anticlockwise at the speed of 40-60 rpm, enabling the processor cylinder (8) to move longitudinally, enabling the second belt (3) to move downwards when the first belt (1) moves upwards, and soaking for 4-6 min;
seventhly, taking down the processor cylinder body (8) from the multifunctional electric support, vertically placing the processor cylinder body in a mode that a sealing silica gel cap (5) is arranged on the multifunctional electric support, opening a cleaning liquid stop valve (14), removing disinfected alcohol, injecting sterile normal saline into the processor cylinder body (8) through the sealing silica gel cap (5), and sealing the processor cylinder body (8); the volume ratio of the sterile normal saline to the processor cylinder (8) is 1 (16-40);
placing a connecting pipe (6) and a cleaning liquid guide pipe (12) on arc-shaped parts of a first belt (1) and a second belt (3) of the multifunctional electric support respectively in a transverse mode of a processor cylinder (8); starting the electric controller (4), enabling the processor cylinder (8) to rotate anticlockwise at the speed of 40-60 rpm, enabling the processor cylinder (8) to move longitudinally, enabling the second belt (3) to move downwards when the first belt (1) moves upwards, and washing for 4-6 min;
ninthly, taking down the processor cylinder (8) from the multifunctional electric support, vertically placing the processor cylinder in an upper form by using a sealing silica gel cap (5), opening the cleaning liquid flow stopping valve (14), removing the cleaning liquid, and closing the cleaning liquid flow stopping valve (14);
secondly, crushing the placenta: a base (15) of the placenta cutting base replaces a conical treatment fluid receiver (10) of a cleaning fluid receiver and is in threaded connection with a processor cylinder (8) of a placenta treatment device, sterile normal saline at 4 ℃ is injected into the processor cylinder (8) through a sealed silica gel cap (5), then the base (15) and an electromagnetic stirrer (2) with a platform are placed on a multifunctional electric bracket in a surface contact mode, the electromagnetic stirrer (2) with the platform is started, a magnetic cutting knife (16) is driven to rotate, the rotating speed is 80-100 rpm, and the treatment is carried out for 5-6 min; the volume ratio of the sterile physiological saline at the temperature of 4 ℃ to the processor cylinder (8) is 1 (80-100);
thirdly, collecting placenta hematopoietic stem cells:
firstly, a conical collection liquid receiver (17) of a hematopoietic cell/tissue collector replaces a base (15) of a placenta shearing base to be in threaded connection with a processor barrel (8) of a placenta processing device, a closed silica gel cap (5) is vertically arranged in an upper mode, a collection liquid stop valve (21) is opened, a single cell suspension rich in hematopoietic stem cells is collected, and the collection liquid stop valve (21) is closed;
injecting placenta tissue enzymolysis liquid into a processor cylinder (8) through a closed silica gel cap (5), sealing the processor cylinder (8), and then respectively placing a connecting pipe (6) and a collected liquid guide pipe (19) on arc-shaped parts of a first belt (1) and a second belt (3) of the multifunctional electric bracket in a transverse mode of the processor cylinder (8); starting an electric controller (4), enabling a processor cylinder (8) to rotate anticlockwise at a speed of 40-60 rpm, enabling the processor cylinder (8) to move longitudinally, enabling a second belt (3) to move downwards when a first belt (1) moves upwards, processing for 10-15 min, taking down the processor cylinder (8) from a multifunctional electric bracket, vertically placing the processor cylinder in a mode that a closed silica gel cap (5) is arranged upwards, opening a collecting liquid stop valve (21), and collecting the single cell suspension rich in hematopoietic stem cells; the placenta tissue enzymolysis liquid is characterized in that a solvent in the placenta tissue enzymolysis liquid is PBS buffer solution, solutes in the placenta tissue enzymolysis liquid are low-molecular-weight heparin sodium injection, I-type collagenase, II-type collagenase, IV-type collagenase and trypsin-EDTA, the concentration of the low-molecular-weight heparin sodium injection is (2-30) IU/mL, the concentration of the I-type collagenase is (0.05-0.5) mg/mL, the concentration of the II-type collagenase is (0.05-0.5) mg/mL, the concentration of the IV-type collagenase is (0.05-0.5) mg/mL, and the concentration of the trypsin-EDTA is (0.05-0.25) mg/mL; the volume ratio of the placenta tissue enzymolysis liquid to the processor barrel (8) is 1 (20-40);
injecting placenta tissue enzymolysis liquid into a processor cylinder (8) through a closed silica gel cap (5), sealing the processor cylinder (8), and then respectively placing a connecting pipe (6) and a collected liquid guide pipe (19) on arc-shaped parts of a first belt (1) and a second belt (3) of the multifunctional electric bracket in a transverse mode of the processor cylinder (8); starting an electric controller (4), enabling a processor cylinder (8) to rotate anticlockwise at a speed of 40-60 rpm, enabling the processor cylinder (8) to move longitudinally, enabling a second belt (3) to move downwards when a first belt (1) moves upwards, processing for 10-15 min, taking down the processor cylinder (8) from a multifunctional electric bracket, vertically placing the processor cylinder in a mode that a closed silica gel cap (5) is arranged upwards, opening a collecting liquid stop valve (21), and collecting the single cell suspension rich in hematopoietic stem cells; the placenta tissue enzymolysis liquid is characterized in that a solvent in the placenta tissue enzymolysis liquid is PBS buffer solution, solutes in the placenta tissue enzymolysis liquid are low-molecular-weight heparin sodium injection, I-type collagenase, II-type collagenase, IV-type collagenase and trypsin-EDTA, the concentration of the low-molecular-weight heparin sodium injection is (2-30) IU/mL, the concentration of the I-type collagenase is (0.05-0.5) mg/mL, the concentration of the II-type collagenase is (0.05-0.5) mg/mL, the concentration of the IV-type collagenase is (0.05-0.5) mg/mL, and the concentration of the trypsin-EDTA is (0.05-0.25) mg/mL; the volume ratio of the placenta tissue enzymolysis liquid to the processor barrel (8) is 1 (20-40);
injecting PBS buffer solution into the processor cylinder (8) through the sealing silica gel cap (5), sealing the processor cylinder (8), and respectively placing the connecting pipe (6) and the collected solution guide pipe (19) on the arc-shaped parts of the first belt (1) and the second belt (3) of the multifunctional electric bracket in a manner that the processor cylinder (8) is transversely placed; starting the electric controller (4) to enable the processor cylinder (8) to rotate anticlockwise at the speed of 40-60 rpm for 4-6 min; taking down the processor cylinder (8) from the multifunctional electric bracket, vertically placing the processor cylinder in a form that a closed silica gel cap (5) is arranged on the processor cylinder, opening a collecting liquid stop valve (21), and collecting the single cell suspension rich in hematopoietic stem cells; the volume ratio of the PBS buffer solution to the processor cylinder (8) is 1 (10-20);
fifthly, merging the single cell suspension rich in hematopoietic stem cells collected in the third step to the fourth step, namely completing the extraction of stem cells separated by placenta tissue enzymolysis;
the multifunctional placenta tissue processing device comprises a multifunctional electric bracket, a placenta processing device, a cleaning fluid receiver, a placenta shearing base and a hematopoietic cell/tissue collector;
the multifunctional electric support comprises a first belt (1), an electromagnetic stirrer (2) with a platform, a second belt (3) and an electric controller (4), wherein the electromagnetic stirrer (2) with the platform is arranged on the electric controller (4), the first belt (1) and the second belt (3) are arranged on two sides of the electric controller (4), arc-shaped parts are arranged on the first belt (1) and the second belt (3), the first belt (1) and the second belt (3) synchronously rotate in an anticlockwise axial direction, but the first belt (1) and the second belt (3) move up and down in a reverse direction;
the placenta treatment device comprises a sealing silica gel cap (5), a connecting pipe (6), a processor cover (7), a processor barrel (8) and two baffles (9), wherein the connecting pipe (6) is arranged at the top of the processor cover (7), the sealing silica gel cap (5) is arranged at the port of the connecting pipe (6), the lower end of the processor cover (7) is connected with the upper end of the processor barrel (8) through threads, and the two baffles (9) are oppositely arranged on the inner wall of the processor barrel (8);
the cleaning solution receiver comprises a conical treatment solution receiver (10), a 200 micron leakage net (11), a cleaning solution guide pipe (12), a cleaning solution latex pipe (13) and a cleaning solution stop valve (14), wherein the 200 micron leakage net (11) is arranged in the conical treatment solution receiver (10), the cleaning solution guide pipe (12) is arranged at the bottom end of the conical treatment solution receiver (10), the tail end of the cleaning solution guide pipe (12) is hermetically connected with the cleaning solution latex pipe (13), and the cleaning solution latex pipe (13) is provided with the cleaning solution stop valve (14);
the placenta cutting base comprises a base (15) and a magnetic shearing knife (16), the upper end of the base (15) is open, the lower end of the base is closed, and the magnetic shearing knife (16) is arranged at the bottom in the base (15);
the hematopoietic cell/tissue collector comprises a conical collection liquid receiver (17), a 70-micron leakage net (18), a collection liquid guide pipe (19), a collection liquid latex pipe (20) and a collection liquid check valve (21); the 70-micron leakage net (18) is arranged in the conical collection liquid receiver (17), the bottom end of the conical collection liquid receiver (17) is provided with a collection liquid guide pipe (19), the tail end of the collection liquid guide pipe (19) is hermetically connected with a collection liquid latex pipe (20), and the collection liquid latex pipe (20) is provided with a collection liquid check valve (21);
when the multifunctional placenta tissue processing device is used for cleaning placenta, the lower end of a processor cylinder (8) of the placenta processing device is in threaded connection with the upper end of a conical processing liquid receiver (10) of a cleaning liquid receiver, and a connecting pipe (6) and a cleaning liquid guide pipe (12) are respectively arranged on the arc-shaped parts of a first belt (1) and a second belt (3) of a multifunctional electric bracket in a mode that the processor cylinder (8) is transversely placed;
when the multifunctional placenta tissue processing device is used for crushing placenta, the lower end of a processor cylinder (8) of the placenta processing device is connected with the upper end of a base (15) of a placenta shearing base through threads, and the multifunctional placenta tissue processing device is arranged on a multifunctional electric bracket in a mode that the base (15) is in surface contact with an electromagnetic stirrer (2) with a platform;
when the multifunctional placenta tissue processing device is used for collecting the placenta hematopoietic cells/tissues, the lower end of a processor cylinder (8) of the placenta tissue processing device is connected with the upper end of a conical collecting liquid receiver (17) of a hematopoietic cell/tissue collector through threads, and a connecting pipe (6) and a collecting liquid guide pipe (19) are respectively arranged on the arc-shaped parts of a first belt (1) and a second belt (3) of the multifunctional electric bracket in a transverse mode of the processor cylinder (8).
2. The method for extracting hematopoietic stem cells from a placenta using the multifunctional placental tissue processing apparatus according to claim 1, wherein the diameter of the arc portion of the first belt (1) is 20cm, and the diameter of the arc portion of the second belt (3) is 20 cm.
3. The method for extracting hematopoietic stem cells from a placenta using the multifunctional placental tissue processing apparatus according to claim 2, wherein the diameter of the plateau of the electromagnetic stirrer (2) is 20 cm.
4. The method for extracting hematopoietic stem cells in the placenta using the multifunctional placental tissue processing apparatus according to claim 1, wherein the processor cover (7) is internally threaded at a lower end thereof and has a diameter of 20 cm; the upper and lower parts of the processor cylinder body (8) are provided with external threads, the diameter is 20cm, and the height is 25 cm.
5. The method for extracting hematopoietic stem cells in placenta using the multifunctional placental tissue processing device according to claim 4, wherein the conical treatment liquid receiver (10) is internally threaded at an upper end thereof and has a diameter of 20 cm; the upper end of the conical collecting liquid receiver (17) is provided with an internal thread, and the diameter of the conical collecting liquid receiver is 20 cm.
6. The method for extracting hematopoietic stem cells from placenta using the multifunctional placental tissue processing apparatus according to claim 4, wherein the two baffles (9) have a rectangular shape, a length of 10cm and a width of 4 cm.
7. The method for extracting hematopoietic stem cells from a placenta using the multifunctional placental tissue processing apparatus according to claim 4, wherein the base (15) is internally threaded at an upper end thereof and has a diameter of 20 cm.
8. The method for extracting hematopoietic stem cells from placenta using the multifunctional placental tissue processing device according to claim 7, wherein the magnetic shearing knives (16) are criss-crossed and have a length of 8cm and a height of 3 cm.
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