CN116410924A - Method for producing platelets in vitro - Google Patents
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- C12N5/0634—Cells from the blood or the immune system
- C12N5/0644—Platelets; Megakaryocytes
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Abstract
The invention provides a method for producing platelets in vitro, and belongs to the technical field of platelet preparation. According to the invention, by designing a specific induction scheme, the phenomenon of platelet burst secretion is realized by inducing macrophages, and the problems that the platelet production efficiency is low and large-scale production cannot be realized in the prior art are solved. The invention can obtain a large number of platelets in a short time, and the proposal of the invention can lead the platelet concentration of the macrophage in the endocrine of unit volume to reach 450 multiplied by 10 9 above/L, an effective way is provided for the scale production of platelet component blood products.
Description
Technical Field
The invention relates to the technical field of platelet preparation, in particular to a method for producing platelets in vitro.
Background
Platelets are an important component of mammalian blood and play an important role in hemostasis, coagulation, and development of immune inflammation. Thrombocytopenia or dysfunction can cause bleeding to varying degrees in the body and in severe cases can even lead to death of the patient. Patients with thrombocytopenia or dysfunctional diseases are currently treated clinically by the method of platelet infusion; however, with the increasing number of total platelet infusions, platelets obtained from gratuitous blood donations alone do not meet clinical demands, while platelets obtained from gratuitous blood donations are limited in terms of short storage time and susceptibility to contamination by pathogens, and thus safer, reliable sources of platelets and more convenient methods of platelet production are needed.
There are some proposals in the prior art for inducing the generation of platelets in vitro using stem cells, including using peripheral blood hematopoietic stem cells, bone marrow hematopoietic stem cells, umbilical cord blood hematopoietic stem cells, embryonic stem cells, etc., for example, heman-giobalasts/Blast Cells (BCs) derived from human embryo cells (human embryonic stem cell, hESC) such as Lu, etc., as intermediate cells for the generation of megakaryocytes, which are induced to generate megakaryocytes and thus platelets. The research group also discusses a culture system suitable for inducing and differentiating BCs cells into megakaryocytes, adopts a TSI culture medium consisting of Stem-line1, 50ng/mL TPO, 20ng/mL Stem Cell Factor (SCF) and 20ng/mL IL-11 to culture 4-6 d of human fetal Stem megakaryocytes (human ESCmegakaryocytes, hESC-MK), adds the human fetal megakaryocytes into a system consisting of OP9 murine stromal cells, a-MEM, 150 mL/L Fetal Bovine Serum (FBS), 50ng/mL SCF, 100ng/mL TPO and 25/mL heparin sodium, continues to culture 4-8d, detects the expression condition of CD41/CD42b through a flow cytometer, and finds that the culture system is favorable for generating platelets by hESC-MK and verifies that the platelets from hESC-have similar characteristics of platelets in normal human blood. However, even so, ESC-induced platelet production is still subject to a complex in vitro culture system, and it is difficult to achieve high in vitro yield or high efficiency of platelet production.
Although the existing studies can obtain megakaryocytes and platelets, the number of platelets produced by differentiation of each megakaryocyte in vitro is still lower than that in vivo, and thus platelet-scale production cannot be achieved.
Disclosure of Invention
The invention aims to provide a method for producing platelets in vitro, which aims to solve the problems that the production efficiency of platelets is low and large-scale production cannot be realized in the prior art.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for producing platelets in vitro, which comprises the following steps:
amplifying and culturing the initial macrophage to obtain amplified macrophage;
carrying out passage on the amplified macrophages to obtain the macrophages after passage;
inoculating the macrophages after passage to an induction culture medium, culturing for 12-36 hours, adding a compound induction reagent into the induction culture medium, and continuously culturing for more than 12 hours to finish the in vitro production of the platelets;
the induction medium comprises the following components in final concentration:
ai Qubo Pa of 40-60 mug/mL and stem cell factor of 50-100 mug/mL;
the compound induction reagent comprises the following components in final concentration:
50-100 ng/mL of thrombopoietin and 50-100 ng/mL of platelet-derived factor.
Preferably, the induction medium adopts alpha-MEM medium and/or 581 medium as basal medium.
Preferably, the amplification culture adopts an amplification culture medium, and the amplification culture medium comprises the following components:
serum substitutes, glutamine solutions and neea non-essential amino acid solutions;
the serum substitute accounts for 3-8% of the total volume of the amplification culture medium;
the glutamine solution accounts for 0.5-1.5% of the total volume of the amplification culture medium;
the NEAA non-essential amino acid solution accounts for 0.5-1.5% of the total volume of the amplification culture medium.
Preferably, the amplification medium uses an alpha-MEM medium and/or 581 medium as the basal medium.
Preferably, the macrophages after passage are second generation cells.
Preferably, the initial macrophages are derived from placental tissue.
Preferably, the initial macrophage is obtained by a preparation method comprising the steps of:
pretreating placenta tissue to obtain tissue fragments;
digesting and filtering the tissue fragments for 2-5 times, merging the filtrates, and stopping digestion to obtain a cell mixture;
pre-purifying the cell mixture to obtain a dispersion cell liquid;
purifying the dispersion cell liquid to obtain initial macrophage;
the placenta tissue is preserved by adopting placenta preservation solution before pretreatment, and/or the placenta tissue is preserved by adopting a condition of 3-5 ℃ before pretreatment;
the placenta preservation solution takes physiological saline as a solvent, and comprises the following components in final concentration:
3-8% of albumin and 0.5-1.5% of green-streptomycin;
the time of preservation is within 36 hours;
the digestion is carried out by adopting a digestion solution, wherein the digestion solution takes 581 culture medium as a solvent and comprises the following components in final concentration:
0.03-0.08% trypsin substitute and 0.05-0.15 g/mL neutral protease;
the mass volume ratio of the tissue fragments to the digestive juice is 1 g:8-12 mL;
the time of each digestion is 8-12 min;
the number of the filtered meshes is 80-120 meshes.
Preferably, the pretreatment comprises the steps of:
removing large blood vessels and connective tissues in placenta tissues, washing with PBS buffer solution until the placenta tissues are gel white, removing small blood vessels and fiber connection components in the placenta tissues, and shearing the tissues to obtain 1-3 mm 3 Organizing the fragments;
the step of stopping digestion specifically comprises the step of mixing filtrate with a serum substitute, wherein the volume ratio of the filtrate to the serum substitute is 2-5%.
Preferably, the pre-purification comprises a first centrifugation, removal of erythrocytes, a second centrifugation, washing, a third centrifugation and resuspension of cells;
the temperature of the first centrifugation, the second centrifugation and the third centrifugation are independently 3-5 ℃, the rotating speeds of the first centrifugation, the second centrifugation and the third centrifugation are independently 800-1200 g, and the time of the first centrifugation, the second centrifugation and the third centrifugation are independently 3-8 min;
collecting the precipitate after the first centrifugation, the second centrifugation and the third centrifugation are finished, and obtaining a first precipitate, a second precipitate and a third precipitate;
the removing of erythrocytes specifically comprises mixing a first precipitate with an erythrocyte lysate;
an ice bath is accompanied in the process of removing the red blood cells;
the time for removing the red blood cells is 20-40 min;
the washing specifically comprises the steps of adopting PBS buffer solution to wash the second precipitate for 2-5 times;
the resuspended cells specifically comprise a dispersed cell fluid obtained by mixing 581 medium with a third pellet.
Preferably, the purification specifically comprises adding the dispersion cell liquid to the upper layer of the lymphocyte separation liquid, centrifuging, wherein the centrifuged system shows a culture medium layer and a lymphocyte separation liquid layer, and initial macrophages are positioned at the interface between the culture medium layer and the lymphocyte separation liquid layer;
the volume ratio of the dispersion cell liquid to the lymphocyte separation liquid is 1:0.8-1.2;
the temperature of the centrifugation is 3-5 ℃, the rotating speed of the centrifugation is 300-700 g, and the time of the centrifugation is 15-25 min.
The invention has the technical effects and advantages that:
according to the invention, by designing a specific induction scheme, the phenomenon of platelet burst secretion is realized by inducing macrophages, a large number of platelets can be obtained in a short time, and an effective way is provided for the large-scale production of platelet component blood products.
Detailed Description
The invention provides a method for producing platelets in vitro, which comprises the following steps: amplifying and culturing the initial macrophage to obtain amplified macrophage; carrying out passage on the amplified macrophages to obtain the macrophages after passage; and inoculating the macrophages after passage to an induction culture medium, culturing for 12-36 hours, adding a composite induction reagent into the induction culture medium, and continuously culturing for more than 12 hours to finish the in vitro production of the platelets.
In the present invention, the initial macrophage is preferably obtained by a preparation method comprising the steps of: pretreating placenta tissue to obtain tissue fragments; digesting and filtering the tissue fragments for 2-5 times, merging the filtrates, and stopping digestion to obtain a cell mixture; pre-purifying the cell mixture to obtain a dispersion cell liquid; and purifying the dispersion cell liquid to obtain the initial macrophage.
The placenta tissue is preferably preserved by adopting placenta preservation solution before pretreatment, the placenta tissue is preferably selected from the placenta of a self-sufficient delivery month-old infant, and the placenta preservation solution takes physiological saline as a solvent and comprises the following components in final concentration: 3-8% of albumin and 0.5-1.5% of green-streptomycin, wherein the final concentration of albumin in the placenta preservation solution is more preferably 4-6%, and the final concentration of green-streptomycin in the placenta preservation solution is more preferably 0.8-1.2%; the preservation temperature is preferably 3-5 ℃, and more preferably 4 ℃; the holding time is preferably within 36 hours, more preferably within 24 hours; the invention selects the placenta for healthy delivery, adopts low temperature and specific placenta preservation liquid to preserve the placenta, enters the test flow in a short time, and aims to extract macrophages with high activity.
In the present invention, the pretreatment preferably includes the steps of: removing large blood vessels and connective tissues in placenta tissues, washing with PBS buffer solution to be in a gel white state, removing small blood vessels and fiber connection components in the placenta tissues, shearing the tissues to obtain 1-3 mm3 tissue fragments, and preferably performing the step in a sterile environment.
In the present invention, the digestion is preferably performed using a digestion solution, preferably in 581 medium, comprising the following components in final concentration: 0.03-0.08% of trypsin substitute and 0.05-0.15 g/mL of neutral protease, wherein the final concentration of the trypsin substitute in the digestion liquid is preferably 0.04-0.06%, and the final concentration of the neutral protease in the digestion liquid is preferably 0.08-0.12 g/mL; the mass volume ratio of the tissue fragments to the digestive juice is preferably 1 g:8-12 mL, and more preferably 1 g:9-11 mL; in the invention, the time of each digestion is preferably 8-12 min, more preferably 9-11 min; the mesh number of the filtration is preferably 80-120 mesh, more preferably 100 mesh, the digestion is preferably carried out at 23-28 ℃, and the digestion is preferably carried out with stirring so as to make the digestion more complete; the method for stopping digestion specifically comprises the steps of mixing filtrate with a serum substitute, wherein the volume ratio of the filtrate to the serum substitute is 2-5%, and more preferably 3-4%. In the invention, placenta tissue is subjected to enzymolysis by adopting a double-enzyme method, and instead of enzymolysis liquid with strong enzymolysis such as pancreatin, DNase and the like which are conventionally used, placenta tissue is fully digested by adopting two enzymolysis liquids such as pancreatin substitutes and neutral enzymes, so that macrophages can be rapidly extracted without being damaged; and 581 culture medium is adopted as a solvent in the enzymolysis liquid, so that good nutrition supply is provided for cells in the enzymolysis process, and the activity of the cells is ensured.
In the present invention, the pre-purification preferably includes a first centrifugation, removal of erythrocytes, a second centrifugation, washing, a third centrifugation, and resuspension of cells; the temperature of the first centrifugation is preferably 3-5 ℃, more preferably 4 ℃, the rotating speed is preferably 800-1200 g, more preferably 1000g, the time is preferably 3-8 min, more preferably 5min; the temperature of the second centrifugation is preferably 3-5 ℃, more preferably 4 ℃, the rotating speed is preferably 800-1200 g, more preferably 1000g, the time is preferably 3-8 min, more preferably 5min; the temperature of the third centrifugation is preferably 3-5 ℃, more preferably 4 ℃, the rotating speed is preferably 800-1200 g, more preferably 1000g, the time is preferably 3-8 min, more preferably 5min; collecting the precipitate after the first centrifugation, the second centrifugation and the third centrifugation are finished, and obtaining a first precipitate, a second precipitate and a third precipitate; the method comprises the steps of removing red blood cells, preferably mixing a first precipitate and a red blood cell lysate, wherein the addition amount of the red blood cell lysate is preferably 20-30 times that of the first precipitate; preferably, the process of removing red blood cells is accompanied by an ice bath; the time for removing the red blood cells is preferably 20-40 min, more preferably 25-35 min; the washing is preferably carried out for 2-5 times on the second precipitate by adopting PBS buffer solution; the resuspended cells are preferably mixed with a 581 medium and a third precipitate to obtain a dispersed cell solution, and the cell concentration in the dispersed cell solution is preferably 1-2×10 5 cells/mL。
In the invention, the purification is preferably carried out by adding the dispersion cell liquid to the upper layer of the lymphocyte separation liquid, centrifuging, and the centrifuged system shows a culture medium layer and a lymphocyte separation liquid layer, wherein initial macrophages are positioned at the interface between the culture medium layer and the lymphocyte separation liquid layer, and the lymphocyte separation liquid is preferably Ficoll diatrizing meglumine lymphocyte separation liquid; the volume ratio of the dispersion cell liquid to the lymphocyte separation liquid is preferably 1:0.8-1.2, and more preferably 1:1; the temperature of centrifugation is preferably 3-5 ℃, more preferably 4 ℃, the rotating speed of centrifugation is preferably 300-700 g, more preferably 400-600 g, the time of centrifugation is preferably 15-25 min, more preferably 18-22 min, the initial macrophages are preferably sucked and transferred to a new container after centrifugation, and are washed and washed by 581 medium, and then are centrifuged again for 10min at 500g at 4 ℃, so as to obtain the purified initial macrophages.
In the present invention, the obtained initial macrophages are subjected to an expansion culture, preferably an expansion culture medium comprising the following components: the serum replacement, the glutamine solution and the NEAA non-essential amino acid solution preferably account for 3-8%, more preferably 4-6% of the total volume of the amplification culture medium, the initial concentration of the glutamine solution is 180-200 mM, the glutamine solution is added to the amplification culture medium after 100 mM dilution, and the glutamine solution preferably accounts for 0.5-1.5%, more preferably 0.8-1.2% of the total volume of the amplification culture medium; the NEAA non-essential amino acid solution preferably accounts for 0.5-1.5%, more preferably 0.8-1.2% of the total volume of the amplification culture medium, the amplification culture medium preferably adopts an alpha-MEM culture medium and/or 581 culture medium as a basic culture medium, more preferably the alpha-MEM culture medium and 581 culture medium are mixed for use, and the addition ratio of the alpha-MEM culture medium to the 581 culture medium is preferably 1:1-2; in the present invention, the macrophages after passage are preferably second generation cells, i.e., progeny cells obtained by once passage.
In the invention, the macrophages after passage are inoculated to an induction culture medium for 12-36 hours, preferably 18-24 hours; the induction medium is preferably an alpha-MEM medium and/or 581 medium as a basic medium, and the alpha-MEM medium and 581 medium are preferably mixed for use, and the addition ratio of the alpha-MEM medium to 581 medium is preferably 1:1-2; the induction medium comprises the following components in final concentration: ai Qubo Pa of 40-60 mug/mL and stem cell factor of 50-100 mug/mL, wherein the final concentration of Ai Qubo Pa is more preferably 45-55 mug/mL, and the final concentration of stem cell factor is more preferably 60-70 mug/mL; then adding a compound induction reagent into the induction culture medium, and continuously culturing for more than 12 hours, preferably 18-36 hours, wherein the compound induction reagent comprises the following components in final concentration: 50-100 ng/mL of thrombopoietin and 50-100 ng/mL of platelet-derived factor, wherein the final concentration of the thrombopoietin is more preferably 60-80 ng/mL, and the final concentration of the platelet-derived factor is more preferably 70-90 ng/mL, so that a large amount of platelets can be induced.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1) Preparing placenta preservation solution with physiological saline as solvent, and adding albumin and 1% of green-streptomycin with final concentration for standby;
placenta of a child who had been given birth was obtained in a hospital, washed with a green-streptomycin-containing PBS buffer (penicillin concentration 100U/ml, streptomycin concentration 0.1 mg/ml), then soaked in placenta preservation solution, stored and transported at 4℃and entered into an experimental procedure within 24 hours after delivery.
2) Tissue treatment: digesting the tissue, placing about 100g of placenta tissue on a glass disc, separating the placenta tissue with sterile scissors to remove large blood vessels and connective tissues, placing PBS for fully washing until the placenta tissue is in a gel white state, removing macroscopic small blood vessels and fiber connection components as far as possible, and cutting the tissue to 1-3 mm 3 . Preparing a digestive juice which takes 581 basal medium as a solvent, and adds trypsin substitute (gibco) with the final concentration of 0.05 percent and neutral protease with the concentration of 0.1g/mL for later use; adding 100mL of digestive juice into every 10g of placenta tissue, stirring at constant speed for 10min at room temperature after adding, and sterilizing3 times of digestion, filtering through a 100-mesh cell filter screen after each digestion, and collecting filtrate.
Adding a helios serum substitute with a volume ratio of 3% into the collected filtrate, uniformly mixing to terminate digestion, centrifuging for 10min at 4 ℃ under the condition of 1000g, discarding the supernatant, adding 15mL of precooled erythrocyte lysate, centrifuging for 5min at 4 ℃ under the condition of 1000r/min after ice bath for 30min, discarding the supernatant, washing cells for 3 times by using PBS buffer, centrifuging for 5min at 4 ℃ under the condition of 1000r/min, discarding the supernatant, and adding 4mL 581 basal medium to resuspend the cells to obtain a cell suspension.
3) Purifying and extracting macrophages: 5mL of Ficoll diatrizer-glucosamine lymphocyte separation liquid (density 1.007 g/L) was added to a 15mL centrifuge tube, and 15mL of the cell suspension obtained in the above step was slowly added to the upper layer of lymphocyte separation liquid, and centrifuged at 500g for 20min at 4 ℃. After centrifugation, the fluid was separated into 3 layers, with macrophages at the interface of the medium and lymphocyte separation fluid. Macrophages were transferred to a 15mL centrifuge tube using a pipette, cells were washed by adding 4mL 581 medium, and after washing, centrifuged at 500g for 10min at 4℃and the supernatant was discarded.
4) Cell inoculation:
preparing a proliferation culture medium: taking an alpha MEM basal medium and a 581 basal medium which are mixed in a ratio of 1:1 as basal media, adding a serum replacement (brand: helios; product number: HPCPLCRL 05) with a final volume concentration of 5%, a glutamine solution (Solarbio, G0200) with a final volume concentration of 1% and a NEAA (Gibco manufacturer, product number: 11140050) with a final volume concentration of 200mM and diluting 100 times for later use;
inoculating the cells obtained in step 3) to a proliferation medium.
5）37℃、5%CO 2 After culturing for 48 hours under the condition of 95% humidity, the supernatant is discarded, and a dish of cells is taken for wall attachment rate calculation.
6) And after the cells are fused, carrying out cell passage to obtain second-generation cells.
7) Cell induction for 48h:
preparing an induction culture medium:
taking an alpha MEM basal medium and a 581 basal medium which are mixed in a ratio of 1:1 as basal media, adding Ai Qubo Pa (eltrobopag) with a final concentration of 50 mug/mL and SCF (stem cell factor) with a final concentration of 70 mug/mL for standby;
inoculating the second-generation cells obtained in the step 6) to an induction culture medium, adding 50ng/mL TPO (thrombopoietin) and 100ng/mL PDGF-BB (platelet derived factor) into the culture medium after induction for 24 hours, wherein TPO is purchased from Shanghai zisource biotechnology Co., ltd, product number 4351-10, and PDGF-BB is purchased from Beijing Qiuweiyi technology Co., ltd, product number CYT-242;
8) And (3) flow detection: and (3) carrying out flow detection when the cells grow to reach a fusion degree of 85 percent: cell suspensions were washed twice with PBS containing 10% fbs; cells were separated into two tubes (1.53X106 cells/tube), stained with 200. Mu.L of 1 XPBS-BSA-anti-macrophage antibody (FITC-labeled) in a centrifuge tube, and incubated with 200. Mu.L of 1 XPBS-BSA as control in another centrifuge tube for 45min at room temperature in the absence of light for detection.
Example 2
1) Preparing placenta preservation solution with physiological saline as solvent, and adding albumin with final concentration of 3% and green-streptomycin with final concentration of 0.5% for later use;
placenta of a child who had been given birth was obtained in a hospital, washed with a green-streptomycin-containing PBS buffer (penicillin concentration 100U/ml, streptomycin concentration 0.1 mg/ml), then soaked in placenta preservation solution, stored and transported at 4℃and entered into an experimental procedure within 24 hours after delivery.
2) Tissue treatment: digesting the tissue, placing about 100g of placenta tissue on a glass disc, separating the placenta tissue with sterile scissors to remove large blood vessels and connective tissues, placing PBS for fully washing until the placenta tissue is in a gel white state, removing macroscopic small blood vessels and fiber connection components as far as possible, and cutting the tissue to 1-3 mm 3 . Preparing a digestive juice which takes 581 basal medium as a solvent, and adds trypsin substitute (gibco) with the final concentration of 0.03 percent and neutral protease with the concentration of 0.05g/mL for later use; 80mL of digestive juice is added into every 10g of placenta tissue, the added placenta tissue is stirred at a constant speed for 8min at room temperature after being added, the placenta tissue is digested for 3 times, and after each digestion, the placenta tissue is filtered through a 100-mesh cell filter screen, and filtrate is collected.
Adding a helios serum substitute with a volume ratio of 3% into the collected filtrate, uniformly mixing to terminate digestion, centrifuging for 10min at 4 ℃ under 800g conditions, discarding the supernatant, adding 15mL of precooled erythrocyte lysate, centrifuging for 5min at 4 ℃ under 1000r/min conditions after ice bath for 20min, discarding the supernatant, washing cells for 3 times by using PBS buffer, centrifuging for 5min at 4 ℃ under 1000r/min, discarding the supernatant, and adding 4mL 581 basal medium to resuspend the cells to obtain a cell suspension.
3) Purifying and extracting macrophages: 5mL of Ficoll diatrizer-gluglucosaminyl lymphocyte separation solution (density 1.007 g/L) was added to a 15mL centrifuge tube, 5mL of the cell suspension obtained in the above step was slowly added to the upper layer of lymphocyte separation solution, and the mixture was centrifuged at 500g for 20min at 4 ℃. After centrifugation, the fluid was separated into 3 layers, with macrophages at the interface of the medium and lymphocyte separation fluid. Macrophages were transferred to a 15mL centrifuge tube using a pipette, cells were washed by adding 4mL 581 medium, and after washing, centrifuged at 500g for 10min at 4℃and the supernatant was discarded.
4) Cell inoculation:
preparing a proliferation culture medium: taking an alpha MEM basal medium and a 581 basal medium which are mixed in a ratio of 1:2 as basal media, adding a serum replacement (brand: helios; product number: HPCPLCRL 05) with a final volume concentration of 3%, a glutamine solution (Solarbio, G0200) with a final volume concentration of 1% and NEAA (Gibco manufacturer, product number: 11140050) with a final volume concentration of 0.5% into the mixture and diluting the mixture 100 times;
inoculating the cells obtained in step 3) to a proliferation medium.
5）37℃、5%CO 2 After culturing for 48 hours under the condition of 95% humidity, the supernatant is discarded, and a dish of cells is taken for wall attachment rate calculation.
6) And after the cells are fused, carrying out cell passage to obtain second-generation cells.
7) Cell induction for 48h:
preparing an induction culture medium:
taking an alpha MEM basal medium and a 581 basal medium which are mixed in a ratio of 1:1 as basal media, adding Ai Qubo Pa (eltrobopag) with a final concentration of 50 mug/mL and SCF (stem cell factor) with a final concentration of 70 mug/mL for standby;
inoculating the second generation cells obtained in the step 6) to an induction culture medium, and adding 50ng/mL TPO (thrombopoietin) and 100ng/mL PDGF-BB (platelet derived factor) into the culture medium after 24 hours of induction
8) And (3) flow detection: and (3) carrying out flow detection when the cells grow to reach a fusion degree of 85 percent: cell suspensions were washed twice with PBS containing 10% fbs; cells were separated into two tubes (1.53X106 cells/tube), stained with 200. Mu.L of 1 XPBS-BSA-anti-macrophage antibody (FITC-labeled) in a centrifuge tube, and incubated with 200. Mu.L of 1 XPBS-BSA as control in another centrifuge tube for 45min at room temperature in the absence of light for detection.
Example 3
1) Preparing placenta preservation solution with normal saline as solvent, and adding albumin with final concentration of 8% and blue-streptomycin with final concentration of 1.5% for standby;
placenta of a child who had been given birth was obtained in a hospital, washed with a green-streptomycin-containing PBS buffer (penicillin concentration 100U/ml, streptomycin concentration 0.1 mg/ml), then soaked in placenta preservation solution, stored and transported at 4℃and entered into an experimental procedure within 24 hours after delivery.
2) Tissue treatment: digesting the tissue, placing about 100g of placenta tissue on a glass disc, separating the placenta tissue with sterile scissors to remove large blood vessels and connective tissues, placing PBS for fully washing until the placenta tissue is in a gel white state, removing macroscopic small blood vessels and fiber connection components as far as possible, and cutting the tissue to 1-3 mm 3 . Preparing a digestive juice which takes 581 basal medium as a solvent, and adds trypsin substitute (gibco) with the final concentration of 0.05 percent and neutral protease with the concentration of 0.1g/mL for later use; 120mL of digestive juice is added into every 10g of placenta tissue, the added placenta tissue is stirred at a constant speed for 10min at room temperature after being added, the placenta tissue is digested for 3 times, and after each digestion, the placenta tissue is filtered through a 100-mesh cell filter screen, and filtrate is collected.
Adding a helios serum substitute with a volume ratio of 3% into the collected filtrate, uniformly mixing to terminate digestion, centrifuging for 10min at 4 ℃ under the condition of 1000g, discarding the supernatant, adding 15mL of precooled erythrocyte lysate, centrifuging for 5min at 4 ℃ under the condition of 1000r/min after ice bath for 30min, discarding the supernatant, washing cells for 3 times by using PBS buffer, centrifuging for 5min at 4 ℃ under the condition of 1000r/min, discarding the supernatant, and adding 4mL 581 basal medium to resuspend the cells to obtain a cell suspension.
3) Purifying and extracting macrophages: 5mL of Ficoll diatrizer-gluglucosaminyl lymphocyte separation solution (density 1.007 g/L) was added to a 15mL centrifuge tube, 5mL of the cell suspension obtained in the above step was slowly added to the upper layer of lymphocyte separation solution, and the mixture was centrifuged at 500g for 20min at 4 ℃. After centrifugation, the fluid was separated into 3 layers, with macrophages at the interface of the medium and lymphocyte separation fluid. Macrophages were transferred to a 15mL centrifuge tube using a pipette, cells were washed by adding 4mL 581 medium, and after washing, centrifuged at 500g for 10min at 4℃and the supernatant was discarded.
4) Cell inoculation:
preparing a proliferation culture medium: taking an alpha MEM basal medium and a 581 basal medium which are mixed in a ratio of 1:1 as basal media, adding a serum replacement (brand: helios; product number: HPCPLCRL 05) with a final volume concentration of 8%, a glutamine solution (Solarbio, G0200) with a final volume concentration of 1% and a NEAA (Gibco manufacturer, product number: 11140050) with a final volume concentration of 1% into the mixture and diluting the mixture 100 times;
inoculating the cells obtained in step 3) to a proliferation medium.
5）37℃、5%CO 2 After culturing for 48 hours under the condition of 95% humidity, the supernatant is discarded, and a dish of cells is taken for wall attachment rate calculation.
6) And after the cells are fused, carrying out cell passage to obtain second-generation cells.
7) Cell induction for 48h:
preparing an induction culture medium:
taking an alpha MEM basal medium and a 581 basal medium which are mixed in a ratio of 1:1 as basal media, adding Ai Qubo Pa (eltrobopag) with a final concentration of 60 mug/mL and SCF (stem cell factor) with a final concentration of 100 mug/mL for standby;
inoculating the second generation cells obtained in the step 6) to an induction culture medium, and adding 100ng/mL TPO (thrombopoietin) and 50ng/mL PDGF-BB (platelet derived factor) into the culture medium after 24 hours of induction
8) And (3) flow detection: and (3) carrying out flow detection when the cells grow to reach a fusion degree of 85 percent: cell suspensions were washed twice with PBS containing 10% fbs; cells were separated into two tubes (1.53X106 cells/tube), stained with 200. Mu.L of 1 XPBS-BSA-anti-macrophage antibody (FITC-labeled) in a centrifuge tube, and incubated with 200. Mu.L of 1 XPBS-BSA as control in another centrifuge tube for 45min at room temperature in the absence of light for detection.
Comparative example 1
The only difference from example 1 is that the final concentration of Ai Qubo Pa was replaced by 200. Mu.g/mL.
Comparative example 2
The only difference from example 1 is that the final concentration of stem cell factor was replaced with 200. Mu.g/mL.
Comparative example 3
The difference from example 1 was that the amount of thrombopoietin added was replaced with 200ng/mL.
Comparative example 4
The difference from example 1 was that the amount of thrombopoietin added was replaced with 10ng/mL.
Comparative example 5
The difference from example 1 was only that the amount of platelet-derived factor added was replaced with 10ng/mL.
Comparative example 6
The difference from example 1 was only that the amount of platelet-derived factor added was replaced with 200ng/mL.
Comparative example 7
The difference from example 1 was only that the amount of platelet-derived factor added was replaced with 0ng/mL.
Experimental example 1 PLt quantitative determination
The amount of PLt secreted by macrophages per unit volume was measured using a cytometer and the results are shown in table 1 below:
TABLE 1 detection of the quantity of PLt secreted by macrophages (n.gtoreq.3)
From the above examples, the present invention provides a method for producing platelets, which can make the concentration of platelets secreted by macrophages in unit volume reach 450×10 9 above/L, it was demonstrated that macrophages are capable of secreting a large amount of platelets under the induction system of the present invention.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for producing platelets in vitro comprising the steps of:
amplifying and culturing the initial macrophage to obtain amplified macrophage;
carrying out passage on the amplified macrophages to obtain the macrophages after passage;
inoculating the macrophages after passage to an induction culture medium, culturing for 12-36 hours, adding a compound induction reagent into the induction culture medium, and continuously culturing for more than 12 hours to finish the in vitro production of the platelets;
the induction medium comprises the following components in final concentration:
ai Qubo Pa of 40-60 mug/mL and stem cell factor of 50-100 mug/mL;
the compound induction reagent comprises the following components in final concentration:
50-100 ng/mL of thrombopoietin and 50-100 ng/mL of platelet-derived factor.
2. The method for producing platelets in vitro according to claim 1, wherein said induction medium uses an alpha-MEM medium and/or 581 medium as a basal medium.
3. The method for producing platelets in vitro according to claim 1, wherein said amplification culture uses an amplification medium comprising the following components:
serum substitutes, glutamine solutions and neea non-essential amino acid solutions;
the serum substitute accounts for 3-8% of the total volume of the amplification culture medium;
the glutamine solution accounts for 0.5-1.5% of the total volume of the amplification culture medium;
the NEAA non-essential amino acid solution accounts for 0.5-1.5% of the total volume of the amplification culture medium.
4. The method for producing platelets in vitro according to claim 3, wherein said amplification medium uses alpha-MEM medium and/or 581 medium as a basal medium.
5. The method of claim 1, wherein the macrophages after passage are second generation cells.
6. The method of in vitro platelet production according to claim 1, wherein said initial macrophages are derived from placental tissue.
7. The method of claim 6, wherein the initial macrophage is obtained by a method comprising:
pretreating placenta tissue to obtain tissue fragments;
digesting and filtering the tissue fragments for 2-5 times, merging the filtrates, and stopping digestion to obtain a cell mixture;
pre-purifying the cell mixture to obtain a dispersion cell liquid;
purifying the dispersion cell liquid to obtain initial macrophage;
the placenta tissue is preserved by adopting placenta preservation solution before pretreatment, and/or the placenta tissue is preserved by adopting a condition of 3-5 ℃ before pretreatment;
the placenta preservation solution takes physiological saline as a solvent, and comprises the following components in final concentration:
3-8% of albumin and 0.5-1.5% of green-streptomycin;
the time of preservation is within 36 hours;
the digestion is carried out by adopting a digestion solution, wherein the digestion solution takes 581 culture medium as a solvent and comprises the following components in final concentration:
0.03-0.08% trypsin substitute and 0.05-0.15 g/mL neutral protease;
the mass volume ratio of the tissue fragments to the digestive juice is 1 g:8-12 mL;
the time of each digestion is 8-12 min;
the number of the filtered meshes is 80-120 meshes.
8. The method for producing platelets in vitro according to claim 7, wherein said pretreatment comprises the steps of:
removing large blood vessels and connective tissues in placenta tissues, washing with PBS buffer solution until the placenta tissues are gel white, removing small blood vessels and fiber connection components in the placenta tissues, and shearing the tissues to obtain 1-3 mm 3 Organizing the fragments;
the step of stopping digestion specifically comprises the step of mixing filtrate with a serum substitute, wherein the volume ratio of the filtrate to the serum substitute is 2-5%.
9. The method for producing platelets in vitro according to claim 7 or 8, wherein said pre-purification comprises a first centrifugation, removal of erythrocytes, a second centrifugation, washing, a third centrifugation and re-suspension of cells;
the temperature of the first centrifugation, the second centrifugation and the third centrifugation are independently 3-5 ℃, the rotating speeds of the first centrifugation, the second centrifugation and the third centrifugation are independently 800-1200 g, and the time of the first centrifugation, the second centrifugation and the third centrifugation are independently 3-8 min;
collecting the precipitate after the first centrifugation, the second centrifugation and the third centrifugation are finished, and obtaining a first precipitate, a second precipitate and a third precipitate;
the removing of erythrocytes specifically comprises mixing a first precipitate with an erythrocyte lysate;
an ice bath is accompanied in the process of removing the red blood cells;
the time for removing the red blood cells is 20-40 min;
the washing specifically comprises the steps of adopting PBS buffer solution to wash the second precipitate for 2-5 times;
the resuspended cells specifically comprise a dispersed cell fluid obtained by mixing 581 medium with a third pellet.
10. The method for producing platelets in vitro according to claim 9, wherein said purification comprises in particular adding a dispersion of cells to the upper layer of lymphocyte separation liquid, and centrifuging, the centrifuged system exhibiting a culture medium layer and a lymphocyte separation liquid layer, the initial macrophages being located at the interface between the culture medium layer and the lymphocyte separation liquid layer;
the volume ratio of the dispersion cell liquid to the lymphocyte separation liquid is 1:0.8-1.2;
the temperature of the centrifugation is 3-5 ℃, the rotating speed of the centrifugation is 300-700 g, and the time of the centrifugation is 15-25 min.
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