CN110878283A

CN110878283A - Method for separating placenta source hemopoietic stem cells

Info

Publication number: CN110878283A
Application number: CN201911401892.5A
Authority: CN
Inventors: 边曙光; 张凤宁; 汪国云; 曾智芃; 明凯
Original assignee: Guizhou Broad Cell Cell Biotechnology Co Ltd
Current assignee: Guizhou Carr Cell Biotechnology Co ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-03-13
Anticipated expiration: 2039-12-30
Also published as: CN110878283B

Abstract

The invention belongs to the field of biomedicine, relates to a method for separating hematopoietic stem cells, and particularly relates to a method for separating placenta-derived hematopoietic stem cells. The method comprises the steps of firstly cutting the placenta tissue, washing and filtering, then mobilizing tissue blocks, and separating the hematopoietic stem cells positioned on the inner wall of a blood vessel from the tissue blocks. The method greatly improves the separation and extraction rate of the placenta-derived hematopoietic stem cells, shortens the preparation time, and obtains the hematopoietic stem cells with higher biological activity. The hematopoietic stem cells obtained by the scheme can be used for replacing bone marrow, mobilized peripheral blood and umbilical cord blood hematopoietic stem cells and treating blood diseases and the like.

Description

Method for separating placenta source hemopoietic stem cells

Technical Field

The invention belongs to the field of biomedicine, relates to a method for separating hematopoietic stem cells, and particularly relates to a method for separating placenta-derived hematopoietic stem cells.

Background

Hematopoietic Stem Cells (HSCs) are a group of primitive Hematopoietic cells present in Hematopoietic tissues that can differentiate, proliferate, and further generate blood cells in different blood cell lines. There are four main sources of hematopoietic stem cells: bone marrow hematopoietic stem cells, peripheral blood hematopoietic stem cells, umbilical cord blood hematopoietic stem cells, placental group hematopoietic stem cells. The placenta tissue has a large hematopoietic stem cell content, and hematopoietic stem cell transplantation using the placenta hematopoietic stem cells can effectively solve the problem of insufficient sources of stem cells such as bone marrow hematopoietic stem cells, and is expected to replace bone marrow, mobilized peripheral blood and umbilical cord blood hematopoietic stem cells for the treatment of blood diseases and the like.

In the prior art, a placenta stem cell is mostly obtained from a placenta tissue by adopting a lavage method, and a general operation flow is that a placenta tissue in vitro is taken, a lavage fluid is injected from an umbilical artery, and then the lavage fluid containing hematopoietic stem cells is collected from an umbilical vein. However, the prior art methods have the following problems: in the process of lavage, vascular embolism and blockage in placenta tissues easily occur, the whole lavage process can not be smoothly carried out, stem cells in the placenta tissues can not be fully collected from umbilical veins, the blockage problem can be solved by adding a certain amount of anticoagulant into the lavage fluid in the prior art, but the state of hematopoietic stem cells can be influenced by adding the anticoagulant; in addition, the hematopoietic stem cells in the placenta tissue are partially located in the blood, and a large part of the hematopoietic stem cells are attached to the vascular endothelium, so that it is difficult to completely wash down the hematopoietic stem cells attached to the vascular endothelium by means of lavage.

Disclosure of Invention

The purpose of the present invention is to provide a method for isolating placenta-derived hematopoietic stem cells, which enables isolation of a large number of hematopoietic stem cells from placenta tissue.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a method of isolating placental derived hematopoietic stem cells, comprising the steps of:

s1 preprocessing step: cutting placenta tissue into tissue blocks I; washing the tissue block I with normal saline, and collecting filtrate I and tissue block II;

s2 organization mobilization step: using the mobilization liquid to mobilize the tissue block II to obtain a tissue block III; the components of the mobilizing liquid comprise AMD3100 and a basal medium;

s3 cell collection step: washing the tissue block III with normal saline, and collecting filtrate II; and filtering and centrifuging the filtrate II, and collecting the lower layer cells II.

By adopting the technical scheme, the technical principle is as follows: after the placenta tissue is washed and minced, a filtrate I containing hematopoietic stem cells is obtained by washing and filtering (i.e., the tissue to be treated is placed on a filter screen, and the tissue is washed with a liquid such as physiological saline (usually 1% double antibody-containing physiological saline) to wash the tissue and collect the substance having a small particle size in the tissue into a collection container), and the tissue mass II also contains a large amount of hematopoietic stem cells attached to the vascular endothelium. Tissue mass II was treated with mobilizing fluid (containing mobilizing agent AMD3100) to obtain tissue mass III (i.e., mobilized placental tissue). And then washing and filtering the tissue block III, collecting filtrate II, and filtering and centrifuging the collected filtrate II to obtain lower layer cells II (containing a large amount of hematopoietic stem cells). Among them, AMD3100 is a CXCR4 chemokine receptor antagonist, and can reversibly bind to CXCR4 on hematopoietic stem cells, interfere with binding of CXCR4 to its ligand, block the physiological processes involved by CXCR4, and mobilize hematopoietic stem cells.

Has the advantages that:

(1) the technical scheme overcomes the technical problem that the hemopoietic stem cells in the tissue cannot be sufficiently eluted by the lavage placenta tissue in the prior art. The technical scheme includes that a placenta tissue is divided into small tissue blocks, then the tissue blocks are treated by using an mobilizing liquid containing a mobilizing agent, the migration of hematopoietic stem cells existing in vascular endothelium out of a blood vessel endothelium position is promoted, and then the migrated hematopoietic stem cells are eluted from the tissue by using a flushing and filtering method. The technical scheme can fully obtain the hematopoietic stem cells in the placenta tissues. The process of perfusing the lavage liquid from the artery of the placenta tissue and collecting the lavage liquid from the vein of the placenta tissue is avoided, the blood vessel blockage can not occur, the hematopoietic stem cells can be effectively collected, the use of a large amount of anticoagulant is avoided, and great help is provided for increasing the activity of the hematopoietic stem cells. This technical scheme uses the placental tissue after the mobilization liquid processing is cut apart, can be with the full mobilization of the hemopoietic stem cell that adsorbs at the blood vessel inner wall be the free state, has increased the volume of the hemopoietic stem cell who obtains.

(2) The technical scheme is that the hematopoietic stem cells in the blood of the placenta tissue are collected by flushing and filtering, and then the hematopoietic stem cells which cannot be directly obtained by flushing in the placenta tissue are treated, namely the placenta tissue is treated by using the mobilizing liquid. Compared with the direct treatment of the tissue block I which is not subjected to washing and filtering by using the mobilization liquid, the operation process avoids the influence of the mobilization liquid on the activity of the free hematopoietic stem cells, because the mobilization liquid can influence the activity of the hematopoietic stem cells to a certain extent.

(3) The lavage method in the prior art usually needs more than 8h of lavage time to complete the separation of hematopoietic stem cells, and has long time and low efficiency. However, the adoption of the method, particularly the step of mobilizing the tissue block, can greatly shorten the separation time of the hematopoietic stem cells. Due to the shortened preparation time, the activity of the hematopoietic stem cells is well maintained, the death rate of the hematopoietic stem cells is reduced to a certain extent, and the extraction rate of the hematopoietic stem cells is increased.

Further, the step of collecting S3 cells further comprises: filtering and centrifuging the filtrate I, and collecting the lower layer cells I; and combining the lower layer cell I and the lower layer cell II to obtain combined cells.

By adopting the technical scheme, the lower layer cell I (containing a large amount of hematopoietic stem cells) is obtained after the filtrate I is filtered and centrifuged. And combining the lower layer cell II and the lower layer cell I to obtain combined cells, wherein the combined cells contain all hematopoietic stem cells obtained from the placenta tissues.

Further, the method also comprises the step of S4 removing red blood cells: resuspending the combined cells in S3 with physiological saline, centrifuging, removing supernatant, and adding hydroxyethyl starch solution to obtain a separation system I; centrifuging the separation system I at the centrifugal speed of 50g for 6min, and removing the erythrocyte layer to obtain a separation system II; and (3) centrifuging the separation system II at the centrifugal rotation speed of 400g for 10min, and removing a plasma layer to obtain a nucleated cell suspension.

By adopting the technical scheme, the mixed red blood cells are removed through gradient centrifugation, so that the aim of purifying the hematopoietic stem cells is fulfilled, and the nucleated cell suspension contains the hematopoietic stem cells.

Further comprises an S5 cell freezing step: uniformly mixing the nucleated cell suspension in the S4, putting the mixture into a freezing bag, adding a freezing solution into the freezing bag, sealing the freezing bag, temporarily storing the freezing bag in a refrigerator at 4 ℃, and cooling to-80 ℃ by using a programmed cooling instrument; then placing the freezing bag in a refrigerator at the temperature of minus 80 ℃ for temporary storage; and after the frozen bags are detected to be qualified, finally placing the frozen bags in a liquid nitrogen tank for long-term storage.

By adopting the technical scheme, the hematopoietic stem cells are cryopreserved in multiple steps, so that the cell preservation time can be prolonged, and the activity of the cells during recovery can be improved.

Further, in S2, the mobilization liquid further includes fetal bovine serum; the volume ratio of the fetal calf serum to the basic culture medium is (4-6): 100.

By adopting the technical scheme, the mobilizing liquid has certain influence on the activity of the hematopoietic stem cells, and the hematopoietic stem cells can be protected by using fetal calf serum.

Further, in S2, AMD3100 and the tissue mass II are used in a ratio of (8-12) mg:1 kg.

By adopting the technical scheme, the mobilizing agent with the dosage can fully mobilize the hematopoietic stem cells and simultaneously reduce the influence of the mobilizing agent on the cell activity to the minimum.

Further, in S2, the mobilizing fluid further comprises collagenase type I, wherein the collagenase type I is present in the mobilizing fluid at a concentration of 0.2 to 0.5 mg/ml.

By adopting the technical scheme, the low-concentration collagenase can increase the mobilization effect of the mobilization liquid on the hematopoietic stem cells and increase the amount of the hematopoietic stem cells obtained by separation. The placenta tissue is treated by the collagenase type I, so that the hematopoietic stem cells adsorbed on the vascular endothelium can be separated, and can be more easily and fully eluted to obtain the hematopoietic stem cells adsorbed on the vascular endothelium. It is conventionally thought that the use of digestive enzymes to treat tissues can make the tissues gel (encapsulate cells and make it difficult to separate hematopoietic stem cells), and the resulting cell types are mixed and make it difficult to subsequently separate hematopoietic stem cells. The inventors have attempted to treat placental tissue with a variety of digestive enzymes, all of which have been shown to occur. However, when the collagenase type I is adjusted to a suitable concentration (0.2 to 0.5mg/ml), the mobilizing effect of the mobilizing fluid is enhanced, and more hematopoietic stem cells can be obtained. The concentration is too low, and the collagenase I has no effect on improving the mobilization effect; at too high a concentration, the placenta tissue is digested, a gelatinous tissue is formed, and a large number of cells of different types (placenta tissue cells) are obtained, which brings great difficulty to subsequent filtration, separation and purification. The inventor analyzes that the hematopoietic stem cells and the vascular endothelial cells have weaker structural relation and are just the action target of the collagenase type I, and the hematopoietic stem cells and the vascular endothelial cells can be separated under the action of the collagenase type I with low concentration, so that the mobilization effect is increased.

Further, in S1, before the placenta tissue is minced, the amnion tissue on the surface of the placenta is removed, the surface of the placenta is washed with a 75% alcohol solution, and the placenta tissue is washed.

Adopt above-mentioned technical scheme, handle the surface with alcohol, will take off the most bacterial colony in surface, reduce the loaded down with trivial details operation and the pollution risk of the degerming in later stage.

Further, the method for cleaning the placenta tissue comprises the following steps: flushing the placenta tissue with physiological saline containing double antibody for 5-8 times; the volume of the tissue block I is 0.1-1mm³。

By adopting the technical scheme, the placenta is flushed by the physiological saline containing double antibiotics (penicillin and streptomycin), so that the placenta tissue can be cleaned, and meanwhile, the proliferation of pathogenic bacteria can be inhibited. Cutting the tissue blocks into pieces of 0.1-1mm³During the subsequent washing, filtering and mobilization process, the washing liquid or the mobilization liquid can be fully contacted with the tissue, and the contact area is increased.

Further, in S2, the temperature of the mobilization treatment of the tissue mass II is 37-39 ℃ and the time period of the mobilization treatment is 30-90 min.

By adopting the technical scheme, the temperature can promote the mobilizing liquid to fully mobilize the tissue block II, so that the hematopoietic stem cells in the adsorption state are separated from the vascular endothelium. The mobilization treatment time is too short to detach the hematopoietic stem cells from the vascular endothelium; the mobilization time is too long, so that the activity of hematopoietic stem cells is lost or the hematopoietic stem cells die, the types of the mobilized cells are complex (various cells in the tissues are mobilized to be in a single cell state), and a large amount of reagent consumables and manpower and material resources are needed for purification at the later stage.

Drawings

FIG. 1 shows the result of microscopic examination (20X-ray microscope) of filtrate I in step S1 of example 1.

FIG. 2 shows the result of microscopic examination (20X light microscope) of the cell suspension in step S3(1) in example 1.

FIG. 3 shows the flow results of the lower layer cell I of example 1.

FIG. 4 shows the flow results of the lower layer cells II of example 1.

FIG. 5 is a photomicrograph (20 Xphotoscope) of the cell colonies of example 1.

FIG. 6 shows the flow results of the lower layer cell I of example 2.

FIG. 7 shows the flow results of the lower layer cells II of example 2.

FIG. 8 shows the photo-microscopic examination of the cell colonies of example 2 (20 Xlight).

FIG. 9 shows the flow results of the lower layer cell I of example 3.

FIG. 10 shows the flow results of the lower layer cells II of example 3.

FIG. 11 is a photomicrograph (20 Xphotoscope) of the cell colonies of example 3.

Detailed Description

The following is further detailed by way of specific embodiments:

example 1: examples of isolating placenta-derived hematopoietic Stem cells

S1 preprocessing step:

the placenta is taken out by sterile hemostat and placed in a sterilized stainless steel plate, the surface amniotic tissue is removed, 75% alcohol is used for washing the surface of the placenta, the placenta is transferred to another sterile iron box, and the edge fetal membrane is removed by scissors. The placenta is washed 5-8 times (the washing times are determined according to the cleanness of the placenta surface, in the embodiment, 6 times) by using 2 bottles of 1 percent (mass fraction) of double antibody (double antibody refers to a solution containing penicillin and streptomycin, wherein 100IU/ml of penicillin and 100IU/ml of streptomycin) normal saline every time, the placenta surface is immersed by the washing liquid, and a sterile iron box is replaced after each washing.

Placing the washed placenta in another sterile tray (adding 1 heparin sodium (12500IU/2ml) in advance to prevent coagulation of blood flowing out of placenta tissue), removing residual umbilical cord and amnion, cutting placenta into pieces, placing into a 600ml beaker, adding 1 heparin sodium anticoagulant into the beaker (or pouring blood containing anticoagulant into the tray without adding anticoagulant), cutting placenta tissue into small pieces (tissue piece I, about 0.1-1 mm) with sterilized tissue scissors³)。

The tissue block I is washed and filtered by a 1.5L beaker provided with an 80-mesh filter screen (the tissue block I is placed on the 80-mesh filter screen) until the filtered liquid is clear (the tissue block I needs to be properly stirred by a pipette in the middle, and cells in the middle of the tissue block are sufficiently washed out). This procedure resulted in filtrate I and tissue mass II. The filtrate I was mixed well, and a small amount of the filtrate I was microscopically examined under a light microscope, and the results are shown in FIG. 1.

S2 organization mobilization step:

and (3) filling the tissue block II into a sterile bottle by using a sterilizing funnel, wherein the sterile bottle is filled with a preheating mobilization liquid prepared in advance, the preheating temperature is 37 ℃, and the mobilization liquid comprises AMD3100, fetal calf serum and a basal culture medium (X-VIVO15 serum-free culture medium). The dosage ratio of AMD3100 and the tissue block II is 10mg:1kg, the volume ratio of the basic culture medium and the tissue block II (or the placenta tissue) is 1:1, and the volume ratio of the bovine serum and the basic culture medium is 5: 100. Sealing the sterile bag, placing in a constant temperature oscillator at 37 ℃ at 150rpm, and mobilizing for 60min to obtain a tissue block III.

S3 cell collection step:

(1) filtering the filtrate I with a 200-mesh cell filter screen, weighing and balancing the filtered cell suspension (a small amount of cell suspension is taken for microscopic examination, and the result is shown in figure 2) with a 250ml centrifuge tube, centrifuging at 1500rpm for 10min, discarding the supernatant to obtain the lower layer cell I, and transferring the lower layer cell I into the 250ml centrifuge tube for later use. A small number of cells were counted and the cell count results are shown in Table 4 (i.e., lower layer cell I count results), and the lower layer cells I in the centrifuge tube were examined by flow cytometry, and the results are shown in FIG. 3 (i.e., lower layer cell I flow results).

(2) The tissue block III was washed and filtered with a 1.5L beaker equipped with an 80 mesh screen until the filtered liquid was clear (the tissue block III was properly stirred with a pipette in the middle to thoroughly wash the cells in the middle of the tissue block). After the above treatment is completed, the tissue block is discarded, the cell suspension is filtered by a 200-mesh filter screen, the filtered cell suspension is weighed and balanced by a 250ml centrifuge tube and centrifuged at 1500rpm for 10min, the supernatant is discarded to obtain the lower layer cells II, the lower layer cells II are uniformly mixed and counted (the cell counting result is shown in table 4, namely the counting result of the lower layer cells II), a small amount of cells are taken for flow cytometry detection (the result is shown in fig. 4, namely the flow result of the lower layer cells II), and then the cells are transferred to the previous 250ml centrifuge tube filled with the lower layer cells I for standby, so that merged cells are obtained.

S4 erythrocyte removing step:

adding physiological saline without double antibody into the combined cells, diluting to 250ml, mixing uniformly, centrifuging at 1500rpm for 10min, and removing supernatant. Adding a hydroxyethyl starch solution with the mass fraction of 1/4 volume of the combined cells being 6% into a centrifugal tube, uniformly mixing, centrifuging at the centrifugal speed of 50g for 6min, and removing the erythrocyte layer; and (4) centrifuging again at the rotating speed of 400g for 10min, and removing a plasma layer to obtain a nucleated cell suspension containing the hematopoietic stem cells. The number of cells in the nucleated cell suspension was counted (the cell count results are shown in table 4, i.e., the total cell count results), and a small number of cells were taken for colony experiments and flow cytometry detection, both of which were confirmatory experiments to determine whether the nucleated cell suspension contained hematopoietic stem cells. Colony experiments as shown in table 1 and fig. 5, two groups of colony experiments were performed, each group was provided with two culture dishes, and the results of the colony experiments show that cells in a nucleated cell suspension can form colonies, indicating the proliferation capacity of hematopoietic stem cells. The flow cytometry test results showed that the positive rate of CD34+ was 10.71%, as shown in table 5.

Table 1: colony assay results (A: red line, B-grain line, C: mixed line)

S5 cell freezing step:

uniformly mixing the nucleated cell suspension, injecting the nucleated cell suspension into an umbilical cord blood cryopreservation bag by using a 60ml injector, slowly adding a pre-precooled cryopreservation solution (comprising two components of DMSO and dextran-40, wherein the volume ratio is 1:1) into the umbilical cord blood cryopreservation bag, uniformly mixing while adding, wherein the volume ratio of the cryopreservation solution to the cell suspension is 1:4, cooling by using gauze sprayed with alcohol above the cryopreservation bag, removing bubbles, sealing in a three-stage manner, and placing a sticker into a cryopreservation iron box and placing in a refrigerator at 4 ℃. And (4) freezing and storing the cells by using a program-controlled cooling instrument, temporarily storing the cells in a refrigerator at minus 80 ℃, and transferring the cells to a deep low-temperature liquid nitrogen tank for long-term storage after the results to be detected are qualified.

Example 2: examples of isolating placenta-derived hematopoietic Stem cells

This example is basically the same as example 1, except that in the S2 tissue digestion step, the digestion temperature is 38 ℃ and the digestion time is 30 min; the dosage ratio of AMD3100 and the tissue block II is 8mg:1kg, and the volume ratio of bovine serum to the basic culture medium is 4: 100. The cell count results of steps S3(1) and (2) and S4 are shown in table 4, the flow cytometer test results of step S3 are shown in fig. 6 and 7 (test for the lower layer cell i and the lower layer cell ii, respectively), and the colony assay results of step S4 are shown in table 2 and fig. 8. Colony experiments indicate the proliferation capacity of hematopoietic stem cells. And flow cytometry detection was performed on the cells (cells in the nucleated cell suspension) obtained in step S4, and the flow cytometry detection experiment results showed that the positive rate of CD34+ was 12.46%, as shown in table 5.

Table 2: colony experimental results (A: red line, B line, C: mixed line)

Example 3

This example is basically the same as example 1, except that in the S2 tissue digestion step, the digestion temperature is 39 ℃ and the digestion time is 90 min; the dosage ratio of AMD3100 and the tissue block II is 12mg:1kg, and the volume ratio of bovine serum to the basic culture medium is 6: 100. The cell count results in steps S3(1) and (2) and S4 are shown in table 4, the flow cytometer test results in step S3 are shown in fig. 9 and 10 (test for the lower layer cell i and the lower layer cell ii, respectively), and the colony assay results in step S4 are shown in table 3 and fig. 11. Colony experiments indicate the proliferation capacity of hematopoietic stem cells. And flow cytometry detection was performed on the cells (cells in the nucleated cell suspension) obtained in step S4, and the flow cytometry detection experiment results showed that the positive rate of CD34+ was 11.53%, as shown in table 5.

Table 3: colony assay results (A: red line, B-grain line, C: mixed line)

Example 4

This example is substantially the same as example 1, except that collagenase type I (Invitrogen) was added to the mobilizing fluid at a concentration of 0.2mg/ml in the mobilizing fluid. Among them, the cell count results of steps S3(1) and (2), S4 are shown in table 4, and the flow cytometry of the cells (cells in the nucleated cell suspension) obtained in step S4 is shown in table 5.

Example 5

This example is substantially the same as example 1 except that collagenase type I was added to the mobilizing fluid and the concentration of collagenase type I in the mobilizing fluid was 0.5 mg/ml. Among them, the cell count results of steps S3(1) and (2), S4 are shown in table 4, and the flow cytometry of the cells (cells in the nucleated cell suspension) obtained in step S4 is shown in table 5.

Example 6

This example is substantially the same as example 1 except that collagenase type I was added to the mobilizing fluid and the concentration of collagenase type I in the mobilizing fluid was 0.35 mg/ml. Among them, the cell count results of steps S3(1) and (2), S4 are shown in table 4, and the flow cytometry of the cells (cells in the nucleated cell suspension) obtained in step S4 is shown in table 5.

Comparative example 1

This comparative example is essentially the same as example 1, except that no fetal bovine serum was added to the mobilizing liquid. Among them, the cell count results of steps S3(1) and (2), S4 are shown in table 4, and the flow cytometry of the cells (cells in the nucleated cell suspension) obtained in step S4 is shown in table 5.

Comparative example 2

This comparative example is essentially the same as example 1, except that no AMD3100 was added to the mobilizer. Among them, the cell count results of steps S3(1) and (2), S4 are shown in table 4, and the flow cytometry of the cells (cells in the nucleated cell suspension) obtained in step S4 is shown in table 5.

Comparative example 3

This comparative example is substantially the same as example 1 except that in the tissue mobilization step of S2, the temperature of the mobilization treatment was 50 ℃ and the time was 120 min. Among them, the cell count results of steps S3(1) and (2), S4 are shown in table 4, and the flow cytometry of the cells (cells in the nucleated cell suspension) obtained in step S4 is shown in table 5.

Comparative example 4

This comparative example is substantially the same as example 1, except that in the tissue mobilization step of S2, the temperature of the mobilization treatment was 20 ℃ and the time was 10 min. Among them, the cell count results of steps S3(1) and (2), S4 are shown in table 4, and the flow cytometry of the cells (cells in the nucleated cell suspension) obtained in step S4 is shown in table 5.

Comparative example 5

This comparative example is essentially the same as example 4, except that collagenase type I is present in the mobilizing liquid at a concentration of 1 mg/ml. Among them, the cell count results of steps S3(1) and (2), S4 are shown in table 4, and the flow cytometry of the cells (cells in the nucleated cell suspension) obtained in step S4 is shown in table 5.

Comparative example 6

This comparative example is essentially the same as example 4, except that collagenase type I is present in the mobilizing liquid at a concentration of 4 mg/ml. Since the concentration of collagenase type I in this comparative example was too high, a large amount of cells of the placenta tissue were digested and separated, and the tissue was gelatinous, so that the subsequent step of S3(2) could not be filtered, and thus, the lower layer cell II fraction could not be obtained. This comparative example was subjected to cell counting (results are shown in Table 4, lower layer cell I) and flow cytometry examination (results are shown in Table 5) only for S3 (1).

Comparative example 7

This comparative example is essentially the same as example 4, except that collagenase type I is present in the mobilizing liquid at a concentration of 0.05 mg/ml. Among them, the cell count results of steps S3(1) and (2), S4 are shown in table 4, and the flow cytometry of the cells (cells in the nucleated cell suspension) obtained in step S4 is shown in table 5.

Comparative example 8

This comparative example is essentially the same as example 4, except that instead of collagenase type I, trypsin is used in an amount of 0.5mg/ml in the mobilizing liquid. Among them, the cell count results of steps S3(1) and (2), S4 are shown in table 4, and the flow cytometry of the cells (cells in the nucleated cell suspension) obtained in step S4 is shown in table 5.

Table 4: cell count results of examples and comparative examples

Table 5: flow cytometry test results of examples and comparative examples

Detecting items	Example 1	Example 2	Example 3	Example 4	Example 5	Example 6	Comparative example 1
								CD34 Positive Rate (%)	6.71	5.46	6.53	10.7	9.1	11.9	2.87
Detecting items	Comparative example 2	Comparative example 3	Comparative example 4	Comparative example 5	Comparative example 6	Comparative example 7	Comparative example 8
								CD34Positive rate (%)	3.34	2.16	3.38	2.01	3.03	3.86	2.65

To ensure comparability of the results, the weight of the selected placental tissue in examples 11-6 and comparative examples 1-8 remained approximately the same, 1.50 + -0.25 kg. From the results in tables 4 and 5, it is understood that the number of cells isolated from the placenta in examples 1 to 3 and examples 4 to 6 is large, and the positive rate of CD34+ is high as measured by flow cytometry, indicating that the number of hematopoietic stem cells isolated is large. Examples 4-6 showed higher numbers of cells isolated (mainly the number of cells in the lower layer II) and higher CD34+ positivity than examples 1-3, indicating that more hematopoietic stem cells adsorbed to the vascular endothelium were mobilized after a small amount of collagenase type I was added to the mobilizing fluid, thereby increasing the number of cells isolated and the CD34+ positivity. In comparative example 1, fetal bovine serum was not added to the mobilizing liquid, the protective effect of fetal bovine serum on hematopoietic stem cells was lacking, the hematopoietic stem cells were poorly active, and partial death occurred. Therefore, the number of the separated lower layer cells II is small, and the positive rate of CD34+ is low. In comparative example 2, AMD3100, a mobilizing agent, was not added to the mobilizing solution, and the number of isolated lower layer cells II was small, and the CD34+ positive rate was low. In comparative example 3, the temperature of the mobilization treatment was too high and the time was too long, resulting in poor hematopoietic stem cell activity and most of the death occurred. Therefore, the number of the separated lower layer cells II is small, and the positive rate of CD34+ is low. In comparative example 4, the temperature of the mobilization treatment was low and the time was too short, resulting in insufficient mobilization of hematopoietic stem cells. Therefore, the number of the separated lower layer cells II is small, and the positive rate of CD34+ is low. In comparative example 5, collagenase type i concentration was higher and some of the placental tissue cells were digested, resulting in higher total cell ratio obtained by separation, but the CD34+ positive rate was decreased, indicating that more heterocytes were separated. In comparative example 6, collagenase type i concentration was very high, and since collagenase type i concentration in this comparative example was too high, a large amount of placental tissue cells were digested and separated, and the tissue was gelatinous, so that the subsequent step of S3(2) could not be filtered, and thus, the lower layer cell fraction ii could not be obtained. In comparative example 7, the collagenase type I concentration was too low and was not significantly different from examples 1-3. In comparative example 8, using another enzyme (trypsin) that digests tissue, it was found that trypsin did not have an effect of assisting mobilization of hematopoietic stem cells. The unexpected effect of using type I collagenase in this protocol is that low concentrations of type I collagenase have an effect in assisting mobilization of hematopoietic stem cells.

The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

Claims

1. A method for isolating placenta-derived hematopoietic stem cells, comprising the steps of:

2. The method of claim 1, wherein the step of collecting S3 cells further comprises: filtering and centrifuging the filtrate I, and collecting the lower layer cells I; and combining the lower layer cell I and the lower layer cell II to obtain combined cells.

3. The method of claim 2, further comprising the step of S4 removing red blood cells: resuspending the combined cells in S3 with physiological saline, centrifuging, removing supernatant, and adding hydroxyethyl starch solution to obtain a separation system I; centrifuging the separation system I, and removing a red blood cell layer to obtain a separation system II; and (4) centrifuging the separation system II, and removing a plasma layer to obtain a nucleated cell suspension.

4. The method for isolating placental derived hematopoietic stem cells according to claim 3, further comprising the step of cryopreserving S5 cells: uniformly mixing the nucleated cell suspension in the S4, putting the mixture into a freezing bag, adding a freezing solution into the freezing bag, sealing the freezing bag, temporarily storing the freezing bag in a refrigerator at 4 ℃, and cooling to-80 ℃ by using a programmed cooling instrument; then placing the freezing bag in a refrigerator at the temperature of minus 80 ℃ for temporary storage; and after the frozen bags are detected to be qualified, finally placing the frozen bags in a liquid nitrogen tank for long-term storage.

5. The method of isolating placental derived hematopoietic stem cells according to any one of claims 1-4, wherein in S2 the mobilizing fluid further comprises fetal bovine serum; the volume ratio of the fetal calf serum to the basic culture medium is (4-6): 100.

6. The method of claim 5, wherein said AMD3100 and said tissue mass II are present in an amount of (8-12) mg:1kg in S2.

7. The method of claim 6, wherein said mobilizing fluid further comprises collagenase type I at a concentration of 0.2 to 0.5mg/ml in said mobilizing fluid at S2.

8. The method of claim 5, wherein in step S1, before the placenta tissue is minced, the amnion tissue on the surface of the placenta is removed, the surface of the placenta is washed with 75% ethanol solution, and the placenta tissue is washed.

9. The method of claim 8, wherein the placental tissue is washed by: flushing the placenta tissue with physiological saline containing double antibody for 5-8 times; the volume of the tissue block I is 0.1-1mm³。

10. The method of claim 5, wherein the temperature of mobilizing tissue block II in S2 is 37-39 ℃ and the length of mobilizing is 30-90 min.