CN110903952B - Method for separating and purifying placenta blood and resuscitating by using protective liquid and placenta extruder - Google Patents
Method for separating and purifying placenta blood and resuscitating by using protective liquid and placenta extruder Download PDFInfo
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- 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
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
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- A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
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- A—HUMAN NECESSITIES
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Abstract
A method for separating and purifying placental blood and resuscitating by using a protective liquid and a placenta extruder relates to a method for separating and purifying placental blood and resuscitating by using a protective liquid and a placenta extruder. The invention aims to solve the problems of difficult separation and extraction of placenta blood, low activation rate of nucleated cells, few cell numbers, easy pollution in the preparation process, reduced cell quality in the recovery process and the like of the conventional method, adopts the placenta extruder and adds the protective solution to fully extrude the blood in the placenta, and then ensures that the total cell number of the nucleated cells can reach 15 x 10 by the high-efficiency separation and purification method 8 The survival rate is above 95%, the clone formation is obvious, the phenotype flow detection CD34 reaches above 0.3%, the pollution rate is controlled within 1%, the survival rate after recovery is above 90%, the phenotype flow detection CD34 has the cloning capacity and can reach above 0.3%, and the sterility test is negative. The invention is applied to the field of placenta hematopoietic stem cell preparation.
Description
Technical Field
The invention relates to a method for separating and purifying placental blood and resuscitating by using a protective liquid and a placenta extruder.
Background
The placenta-derived hematopoietic stem cells are a group of primitive hematopoietic cells present in placenta tissue, and the hematopoietic stem cells are highly undifferentiated cells, which can be said to be primitive cells of all blood cells (most of them are immune cells).
The transplantation can treat 75 lethal diseases such as malignant hematopathy, partial malignant tumor, partial hereditary disease, etc. such as acute leukemia, chronic leukemia, myelodysplastic syndrome, hematopoietic stem cell disease, myeloproliferative disease, lymphoproliferative disease, macrophage disease, hereditary metabolic disease, histiocyte disease, hereditary erythrocyte disease, hereditary immune system disease, hereditary platelet disease, plasma cell disease, thalassemia, non-hematological malignancy, acute radiation disease, etc.
At present, placenta blood is not thoroughly extracted during separation and extraction, and a large amount of nucleated cells are lost during preparation and freezing storage, and the cell number is 5-10 x 10 8 And the preparation time is long, the physiological saline is insufficient for protecting cells, so that a large number of nucleated cells die, the living rate is 60-80%, the whole preparation process is basically in an open state, the pollution rate is 15-20%, and the quality is obviously reduced after freezing and recovery.
Disclosure of Invention
The invention aims to solve the problems of difficult separation and extraction of placenta blood, low survival rate of nucleated cells, small cell number, long preparation time, low phenotype detection value and easy pollution in the preparation process of the traditional method, and provides a method for separating and purifying placenta blood and resuscitating by using a protective liquid and a placenta extruder.
The invention relates to a placenta extruder, which comprises an extrusion rod, a fixed rod, an extrusion cylinder, an extrusion disc, a compression-resistant net, a filter screen and a fixed seat, wherein the fixed rod is arranged on the extrusion disc; the extrusion rod is in threaded connection with the fixed rod, and the lower end of the extrusion rod is connected with the extrusion disc; in the extrusion process, the extrusion disc stretches into the extrusion cylinder; a compression-resistant net and a filter screen are arranged below the inside of the extrusion cylinder, and the compression-resistant net is arranged above the filter screen; the bottom of the extrusion cylinder is conical, and a pipeline connecting port is formed at the bottom end; the fixing seat is fixedly connected to the upper part of the outer wall of the extrusion cylinder, and the fixing rod is inserted into the fixing seat and is fixed through the fixing bolt.
The fixing seat of the placenta extruder is provided with the fastening bolt, the inserted fixing rod is fixed through the fastening bolt, the bolt is unscrewed, and the extruding disc can be pulled out of the extruding cylinder by pulling the fixing rod, so that impurities such as blood coagulation and tissues in placenta or hematopoiesis can be conveniently put in and removed. When the placenta tissue block is used, the washed placenta tissue block is put on the compression-resistant net in the extrusion barrel, the extrusion rod is rotated to drive the extrusion disc to extrude placenta tissue, blood in the placenta is filtered by the filter net and is discharged into the blood collection bag through the pipeline connection port, so that the placenta blood is fully extruded out, and the pollution risk and the preparation time are reduced.
The invention relates to a method for separating and purifying placental blood by using a protective liquid and a placenta extruder, which comprises the following steps:
1. adding 100 Xdiab into physiological saline as a cleaning solution, wherein the final volume concentration of the 100 Xdiab in the cleaning solution is 1%;
2. weighing sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, L-malic acid and sodium hydroxide, adding into water for injection, stirring to dissolve completely, adjusting pH to 7.0-7.2, and adding low molecular heparin sodium injection and dextran to prepare a protective solution; wherein the final concentration of the low molecular heparin sodium in the protective solution is 25-35IU/mL, the final concentration of the dextran is 0.025g/mL, and the mass ratio of sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, L-malic acid and sodium hydroxide is (3-4); (0.15-0.2): (0.2-0.3): (0.1-0.2): (0.15-0.2); (0.3-0.4); (0.1-0.2); the mass volume ratio of the sodium chloride to the water for injection is (3-4) g (490-530) mL;
3. washing placenta with washing liquid for 2-3 times, soaking placenta for 4-10min, taking out placenta, washing placenta with protective liquid for 1-2 times, cutting placenta into small pieces, placing on compression-resistant net in placenta extruder, connecting sample outlet of extruder with blood collecting bag, pouring 150-200mL protective liquid into placenta extruder, rotating extrusion rod, driving extrusion plate, extruding placenta, extruding blood into blood collecting bag, shaking table mixing for 4-10min, and connecting blood collecting bag with triple bag, wherein the triple bag comprises transfer bag, blood plasma bag and freezing bag; transferring the sample in the blood sampling bag into a transfer bag, sampling and counting, and detecting the total volume of red blood cells in the transfer bag;
if the total volume of the red blood cells in the transfer bag is larger than x mL in the detection result; adding hydroxyethyl starch according to 20% of the total volume of the sample in the transfer bag, uniformly mixing for 4-10min by a shaking table, centrifuging for the first time, discharging erythrocytes in the transfer bag into a blood collection bag after centrifuging, discharging the volume of erythrocytes = the total volume of erythrocytes in the transfer bag-x mL, centrifuging for the second time, discharging plasma in the transfer bag into a plasma bag after centrifuging, and discharging the volume of plasma = the total volume of the sample in the transfer bag-x mL-discharging the volume of erythrocytes;
if the total volume of red blood cells in the transfer bag is smaller than xmL in the detection result, directly centrifuging, and discharging plasma in the transfer bag into a plasma bag after centrifuging, wherein the plasma discharge volume=the total volume of samples in the transfer bag-x mL; wherein x mL is the target volume of cryopreserved placental blood;
4. pre-cooling the transfer bag processed in the step three in a refrigerator, adding frozen stock solution into placenta blood of the transfer bag, fully and uniformly mixing, transferring to the frozen stock bag, taking the frozen stock bag off from the triple bag, sealing, balancing for 4-10min in the refrigerator, transferring to a refrigerator at-20 ℃ for 1-2 hours, transferring to a refrigerator at-80 ℃ for 4-6 hours, and finally transferring to liquid nitrogen at-196 ℃ for freezing; the volume ratio of placenta blood to frozen stock solution in the transfer bag is (5-7): 1.
The method for recovering the placental blood comprises the following steps: balancing frozen placenta blood in a liquid nitrogen tank for 1-3min, transferring to a water bath kettle at 37 ℃ for resuscitation, taking out after thawing, transferring to a centrifuge tube with a protective agent, centrifuging at 1100-1400g and 8-12 ℃ for 5-8min at the volume ratio of the frozen placenta blood to the protective agent of 1 (8-11), and removing the supernatant after centrifuging; wherein the protective agent is a compound electrolyte solution containing human serum albumin and dextran, the final concentration of the human serum albumin in the protective agent is 1%, and the final concentration of the dextran in the protective agent is 5%.
The invention uses self-prepared protective liquid, 3-4g of sodium chloride, 0.15-0.2g of potassium chloride, 0.2-0.3g of calcium chloride dihydrate, 0.1-0.2g of magnesium chloride hexahydrate, 0.15-0.2g of sodium acetate trihydrate, 0.3-0.4g of L-malic acid and 0.1-0.2g of sodium hydroxide are weighed, added into 490-530mL of water for injection, stirred until the sodium chloride is completely dissolved, the pH value is regulated to 7.0-7.2, then low-molecular heparin sodium injection and dextran are added, the concentration of the low-molecular heparin sodium is 25-35IU/mL, the final concentration of the dextran is 0.025g/mL, and the protective liquid is prepared, so that the activity rate, the non-caking and the cloning capacity of nucleated cells of a sample can be ensured in the preparation and freezing process, and the detection of a phenotype CD34 is more than 0.3%.
The beneficial effects of the invention are as follows:
the invention uses the placenta extruder to fully extrude the blood in the placenta, and the protective liquid plays a role in nucleated cellsThe protection effect is fixed, and then the total cell number of the nucleated cells in the prepared placenta blood can reach 15 x 10 through a high-efficiency separation and purification method 8 The method has the advantages that the activity rate is more than 95%, the clone formation is obvious, the phenotype flow type detection CD34 reaches more than 0.3%, the pollution rate is controlled within 1%, the preparation time is controlled within 2.5 hours, the frozen stock solution is added to cool to-80 ℃, the frozen stock solution is stored in liquid nitrogen for 6 months and then is recovered, the activity rate is more than 90% after recovery by a new recovery method, the phenotype flow type detection CD34 has the cloning capacity and can reach more than 0.3%, and the sterility test is negative.
Drawings
FIG. 1 is a schematic view of the structure of a placenta extruder of the present invention;
FIG. 2 is a schematic view of a compression resistant mesh and screen of the placenta extruder of the present invention;
FIG. 3 is a graph showing the differentiation of clone colonies before cryopreservation of nucleated cells in isolated and purified placental blood according to example 1;
FIG. 4 is a graph showing the differentiation of the recovered clone colonies after cryopreservation of nucleated cells in isolated and purified placental blood according to example 1.
Detailed Description
The first embodiment is as follows: the placenta extruder of the embodiment comprises an extrusion rod 1, a fixed rod 2, an extrusion cylinder 3, an extrusion disk 4, a compression-resistant net 5, a filter screen 6 and a fixed seat 8; the extrusion rod 1 is in threaded connection with the fixed rod 2, and the lower end of the extrusion rod 1 is connected with the extrusion disc 4; in the extrusion process, the extrusion disc 4 stretches into the extrusion cylinder 3; a compression-resistant net 5 and a filter screen 6 are arranged below the inside of the extrusion cylinder 3, and the compression-resistant net 5 is arranged above the filter screen 6; the bottom of the extrusion cylinder 3 is conical, and the bottom end is provided with a pipeline connecting port 7; the fixing seat 8 is fixedly connected to the upper part of the outer wall of the extrusion cylinder 3, and the fixing rod 2 is inserted into the fixing seat 8 and is fixed through a fastening bolt.
In this embodiment, the diameter of the extrusion disk 4, the compression-resistant net 5 and the filter screen 6 are equal.
The fixing seat 8 of the placenta extruder in the embodiment is provided with a fastening bolt, the fixing rod 2 which is fixedly inserted through the fastening bolt is unscrewed, the extruding disc 4 can be pulled out of the extruding cylinder 3 through pulling the fixing rod 2, and impurities such as blood coagulation and tissues in placenta or hematopoietic tissues can be conveniently placed in or removed from the extruding cylinder. When in use, the cleaned placenta tissue block is put on the compression-resistant net 5 in the extrusion cylinder 3, the extrusion rod 1 is rotated to drive the extrusion plate 4 to extrude the placenta tissue, blood in the placenta is filtered by the filter screen 6 and is discharged into the blood collection bag through the pipeline connection port 7.
The placenta extruder and the protective liquid of the embodiment are adopted to fully extrude the blood in the placenta and protect the cells, and then the high-efficiency separation, purification and recovery method is adopted to ensure that the total cell number of the nucleated cells in the placenta blood can reach 15 x 10 8 The method has the advantages that the activity rate is above 95%, the clone formation is obvious, the phenotype flow type detection CD34 reaches above 0.3%, the pollution rate is controlled within 1%, the freezing solution is added for gradient cooling to-80 ℃, the freezing solution is transferred into liquid nitrogen for storage for 6 months and then resuscitated, the activity rate after resuscitation is above 90%, the clone capacity is achieved, the phenotype flow type detection CD34 can reach above 0.3%, and the sterility test is negative.
The second embodiment is as follows: the first difference between this embodiment and the specific embodiment is that: the extrusion cylinder 3, the extrusion disk 4 and the compression-resistant net 5 are all made of stainless steel. The other is the same as in the first embodiment.
And a third specific embodiment: this embodiment differs from the first or second embodiment in that: the filter screen 6 is a 100-mesh filter screen. The other embodiments are the same as those of the first or second embodiment.
The specific embodiment IV is as follows: the method for separating and purifying placental blood by using the protective liquid and the placenta extruder in the embodiment is carried out according to the following steps:
1. adding 100 Xdiab into physiological saline as a cleaning solution, wherein the final volume concentration of the 100 Xdiab in the cleaning solution is 1%;
2. weighing sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, L-malic acid and sodium hydroxide, adding into water for injection, stirring to dissolve completely, adjusting pH to 7.0-7.2, and adding low molecular heparin sodium injection and dextran to prepare a protective solution; wherein the final concentration of the low molecular heparin sodium in the protective solution is 25-35IU/mL, the final concentration of the dextran is 0.025g/mL, and the mass ratio of sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, L-malic acid and sodium hydroxide is (3-4); (0.15-0.2): (0.2-0.3): (0.1-0.2): (0.15-0.2); (0.3-0.4); (0.1-0.2); the mass volume ratio of the sodium chloride to the water for injection is (3-4) g (490-530) mL;
3. washing placenta with washing liquid for 2-3 times, soaking placenta for 4-10min, taking out placenta, washing placenta with protective liquid for 1-2 times, cutting placenta into small pieces, placing on compression-resistant net 5 in placenta extruder, connecting sample outlet 7 of extruder with blood collecting bag, pouring 150-200mL protective liquid into placenta extruder, rotating extrusion rod 1, driving extrusion plate 4, extruding placenta, extruding blood into blood collecting bag, shaking table mixing for 4-10min, and connecting blood collecting bag with triple bag, wherein the triple bag comprises transfer bag, blood plasma bag and freezing bag; transferring the sample in the blood sampling bag into a transfer bag, sampling and counting, and detecting the total volume of red blood cells in the transfer bag;
if the total volume of the red blood cells in the transfer bag is larger than x mL in the detection result; adding hydroxyethyl starch according to 20% of the total volume of the sample in the transfer bag, uniformly mixing for 4-10min by a shaking table, centrifuging for the first time, discharging erythrocytes in the transfer bag into a blood collection bag after centrifuging, discharging the volume of erythrocytes = the total volume of erythrocytes in the transfer bag-x mL, centrifuging for the second time, discharging plasma in the transfer bag into a plasma bag after centrifuging, and discharging the volume of plasma = the total volume of the sample in the transfer bag-x mL-discharging the volume of erythrocytes;
if the total volume of red blood cells in the transfer bag is smaller than xmL in the detection result, directly centrifuging, and discharging plasma in the transfer bag into a plasma bag after centrifuging, wherein the plasma discharge volume=the total volume of samples in the transfer bag-x mL; wherein x mL is the target volume of cryopreserved placental blood;
4. pre-cooling the transfer bag processed in the step three in a refrigerator, adding frozen stock solution into placenta blood of the transfer bag, fully and uniformly mixing, transferring to the frozen stock bag, taking the frozen stock bag off from the triple bag, sealing, balancing for 4-10min in the refrigerator, transferring to a refrigerator at-20 ℃ for 1-2 hours, transferring to a refrigerator at-80 ℃ for 4-6 hours, and finally transferring to liquid nitrogen at-196 ℃ for freezing; the volume ratio of placenta blood to frozen stock solution in the transfer bag is (5-7): 1.
Fifth embodiment: the fourth difference between this embodiment and the third embodiment is that: 100 Xdiabody is a mixed solution of penicillin and streptomycin. The other is the same as in the fourth embodiment.
Specific embodiment six: the present embodiment differs from the fourth or fifth embodiment in that: the first centrifugation parameters for red blood cell volumes greater than x mL were: the rotating speed is 50-100g, the time is 5-10min, and the temperature is 10 ℃; the parameters for the second centrifugation were: the rotating speed is 500-800g, the time is 15-20min, and the temperature is 10 ℃; when the volume of the red blood cells is smaller than xmL, the parameters of centrifugation are as follows: the rotating speed is 500-800g, the time is 15-20min, and the temperature is 10 ℃. The others are the same as those of the fourth or fifth embodiment.
Seventh embodiment: the present embodiment differs from one of the fourth to sixth embodiments in that: and step four, the transfer bag is placed into a refrigerator with the temperature of 4 ℃ to be precooled for 10min. The others are the same as those of the fourth to sixth embodiments.
Eighth embodiment: the present embodiment differs from one of the fourth to seventh embodiments in that: and step four, the frozen stock solution is prepared from dimethyl sulfone and dextran, wherein the final volume concentration of the dimethyl sulfone in the frozen stock solution is 50%, and the final volume concentration of the dextran is 5%. The others are the same as in one of the fourth to seventh embodiments.
Detailed description nine: the present embodiment differs from one of the fourth to eighth embodiments in that: and fourthly, placing the freezing bag into a refrigerator with the temperature of 4 ℃ for balancing for 5min, transferring into a medical refrigerator with the temperature of-20 ℃ for 1.5 hours, transferring into a refrigerator with the low temperature of-80 ℃ for 5 hours, and finally transferring into liquid nitrogen with the temperature of-196 ℃ for freezing. The others are the same as in one of the fourth to eighth embodiments.
Detailed description ten: the method for recovering placental blood in the embodiment comprises the following steps: balancing frozen placenta blood in a liquid nitrogen tank for 1-3min, transferring to a water bath kettle at 37 ℃ for resuscitation, taking out after thawing, transferring to a centrifuge tube with a protective agent, centrifuging at 1100-1400g and 8-12 ℃ for 5-8min at the volume ratio of the frozen placenta blood to the protective agent of 1 (8-11), and removing the supernatant after centrifuging; wherein the protective agent is a compound electrolyte solution containing human serum albumin and dextran, the final concentration of the human serum albumin in the protective agent is 1%, and the final concentration of the dextran in the protective agent is 5%.
The effect of the invention is verified by the following examples:
example 1: a method for separating and purifying placenta blood by using a protective liquid and a placenta extruder comprises the following steps:
1.5 mL of 100 Xdiabody (penicillin, streptomycin) was added to 500mL of physiological saline to obtain a washing solution;
2. 3g of sodium chloride, 0.15g of potassium chloride, 0.2g of calcium chloride dihydrate, 0.1g of magnesium chloride hexahydrate, 0.15g of sodium acetate trihydrate, 0.3g of L-malic acid and 0.1g of sodium hydroxide are prepared in advance, added into 500ml of water for injection, stirred until the sodium chloride is completely dissolved, the pH value is regulated to 7.0-7.2 by hydrochloric acid or sodium hydroxide, and then the low molecular heparin sodium injection and dextran are added to prepare the protective solution; the final concentration of the low molecular heparin sodium in the protective solution is 25-35IU/mL, and the final concentration of the dextran is 0.025g/mL.
3. Taking out the sterilized tray, hemostatic forceps and tissue shears from the instrument box, clamping the placenta from the sterile bag by using the hemostatic forceps, putting the placenta into the tray, shearing the tissue to remove the umbilical cord, the amniotic membrane, the blood coagulation and the oil impurities on the placenta, washing the placenta for 3 times by using a washing liquid, soaking the placenta for 5 minutes by using the washing liquid, taking out the placenta, washing the placenta for 2 times by using a protective liquid, cutting the placenta into small pieces, putting the small pieces on a compression-resistant net 5 in a placenta extruder, connecting a sample outlet 7 of the extruder with a blood sampling bag, pouring 150mL of protective liquid into the placenta extruder, rotating an extrusion rod 1, driving an extrusion disc 4, extruding the placenta, extruding the blood into the blood sampling bag, uniformly mixing the shaking table for 5 minutes, and connecting the blood sampling bag with a triple bag, wherein the triple bag is divided into a transfer bag, a plasma bag and a freezing bag; transferring the sample in the blood sampling bag into a transfer bag, sampling and counting, and detecting the number of nucleated cells and the total volume of red blood cells in the sample in the transfer bag;
if the total volume of the red blood cells in the transfer bag is larger than 20mL in the detection result; adding hydroxyethyl starch according to 20% of the total volume of the sample in the transfer bag, uniformly mixing for 4-10min by a shaking table, then carrying out first centrifugation (50-100 g, 5-10min and 10 ℃), placing the transfer bag mouth downwards during centrifugation, positioning erythrocytes at the bag mouth position after centrifugation, opening a tube clamp of a triple bag, discharging erythrocytes in the transfer bag into a blood collection bag, discharging erythrocytes volume = total volume of erythrocytes in the transfer bag-20 mL, then carrying out second centrifugation, placing the transfer bag mouth upwards during centrifugation, opening the tube clamp of the triple bag after centrifugation, discharging plasma in the transfer bag into the plasma bag in an extrusion mode, and discharging plasma discharge volume = total volume of the sample in the transfer bag-20 mL-discharge erythrocytes volume;
if the total volume of red blood cells in the transfer bag is smaller than 20mL in the detection result, directly centrifuging (500-800 g, 15-20min and 10 ℃), and discharging the plasma in the transfer bag into a plasma bag after centrifuging, wherein the plasma discharge volume=the total volume of samples in the transfer bag is-20 mL; and (5) fully and uniformly mixing the residual samples in the transfer bag, and sampling and inspecting.
The conventional method for the comparative group is: cleaning with physiological saline as a protective agent without adding any substance, transferring the cleaned blood into a sterile bag in a block shape, squeezing the blood into a tray by hand, pouring the physiological saline into the sterile bag, repeatedly cleaning and squeezing the blood twice, filtering the liquid in the tray by using a 100-mesh stainless steel funnel, transferring the liquid into a 50ml centrifuge tube for centrifugation, recovering the upper plasma, injecting the upper plasma into a sterile detection bottle, recovering the middle white membrane layer into a new centrifuge tube, fully mixing the blood evenly, sampling and delivering the blood to the test tube.
For 15 batches of the conventional preparation method and the new preparation method as comparison groups, the samples were submitted for nucleated cell counting, nucleated cell viability, flow CD34 phenotype detection, clone colony formation, sterility and preparation time statistics, and the results are shown in Table 1.
4. Pre-cooling the transfer bag treated in the third step in a refrigerator, adding frozen stock solution into the transfer bag, fully and uniformly mixing, balancing for 5min in a refrigerator at 4 ℃, transferring into a medical refrigerator at-20 ℃ for 1.5 hours, transferring into a low-temperature refrigerator at-80 ℃ for 5 hours, and finally transferring into liquid nitrogen at-196 ℃ for freezing; the volume ratio of placenta blood to frozen stock solution in the transfer bag is 6:1.
The method for resuscitating the frozen placental blood comprises the following steps: and (3) finding out the sample, taking the sample to a liquid nitrogen tank to balance for 1min, quickly transferring the sample into a water bath kettle at 37 ℃ for resuscitation, taking out the sample in time after the sample is melted, and transferring the sample into a 50ml centrifuge tube with a protective agent, wherein the ratio of the sample to the protective liquid is 1:10. Wherein the protective agent is a compound electrolyte solution containing human serum albumin and dextran, the final concentration of the human serum albumin in the protective agent is 1%, the final concentration of the dextran in the protective agent is 5%, centrifuging at 1300 rpm and 7 min and at 10 ℃, extracting the supernatant after centrifuging, injecting the supernatant into a sterile detection bottle, discarding the rest supernatant, and carrying out detection on the survival rate, phenotype and colony formation and counting of a bottom sample; wherein the supernatant is a protective agent.
The conventional method for the comparative group is: the water bath is adjusted to 37 ℃ in advance, frozen placenta blood is taken out from liquid nitrogen and put into the water bath for 2min, then the placenta blood is transferred into normal saline, centrifugation (1300-1500 rpm, 5-10min and 10 ℃) is carried out, after the completion, a supernatant fluid injection detection bottle is extracted to detect sterility, and the remaining samples are sent to be detected for nuclear cell number, activity rate, clone colony formation and flow CD34 phenotype detection.
And respectively taking 2 tubes of 15 batches of frozen samples, respectively carrying out resuscitating detection by using a conventional resuscitating method and a new resuscitating method, and comparing the nucleated cell number, the activity rate, the clone colony formation, the phenotype detection of the streaming CD34 and the sterile detection. The results of the comparison are shown in Table 2.
The placenta extruder of the embodiment is shown in fig. 1, and comprises an extrusion rod 1, a fixed rod 2, an extrusion cylinder 3, an extrusion disk 4, a compression-resistant net 5, a filter screen 6 and a fixed seat 8; the extrusion rod 1 is in threaded connection with the fixed rod 2, and the lower end of the extrusion rod 1 is connected with the extrusion disc 4; in the extrusion process, the extrusion disc 4 stretches into the extrusion cylinder 3; a compression-resistant net 5 and a filter screen 6 are arranged below the inside of the extrusion cylinder 3, and the compression-resistant net 5 is arranged above the filter screen 6; the bottom of the extrusion cylinder 3 is conical, and the bottom end is provided with a pipeline connecting port 7; the fixed seat 8 is fixedly connected to the upper part of the outer wall of the extrusion cylinder 3, and the fixed rod 2 is inserted into the fixed seat 8 and is fixed through a fastening bolt; wherein a schematic view of the compression-resistant net 5 and the filter screen 6 is shown in fig. 2.
TABLE 1
From table 1, it can be seen that the number, the activity rate, the phenotype CD34 expression, the colony formation and the average value of the placental blood purification by using the placenta extruder and the protective solution are all much higher than those of the conventional preparation method, the preparation time of the novel method is also reduced, the average value of the bacterial infection positive reporting rate of the novel method for the sterile detection of the batch is 0, and the conventional method is 15.9. The new preparation method fully improves the quality of the sample.
TABLE 2
From table 2, it can be seen that the conventional resuscitation method is greatly reduced compared with the sample survival rate, cell number, flow phenotype CD34 detection and clone colony formation before freezing, and the new resuscitation method has a certain effect on sample protection because the descending resuscitation is smaller although the new resuscitation method is reduced compared with the sample before freezing.
As can be seen from table 1, the collection amount of nucleated cells in the placental blood is greatly improved by using the extruder of the embodiment, the preparation time is reduced, the working efficiency is ensured, the placental blood is protected by the protecting liquid, the osmotic pressure of the placental blood cells is ensured, cell aggregation and apoptosis are avoided, the activity rate is relatively improved in the clone colony formation and the streaming CD34 expression, the pollution rate is controlled below 1%, the same batch of cells are compared with the new resuscitation method by using the conventional resuscitation method, the detection quality of the nucleated cells, the cloning capacity detection, the streaming CD34 expression and the like of the sample are better than the conventional resuscitation method by using the effects of the protecting agent in the freezing storage liquid and the resuscitation process, and the cloning capacity is compared with the new resuscitation method by using the new preparation method with no obvious difference, the cloning capacity is relatively strong, and the pollution rate of the sample is controlled below 1% by using the triple bag for separation by using the cleaning liquid in the early stage of the embodiment and the whole process as shown in fig. 3 and 4; the triple bag of this example was purchased from Thermogenesis corporation.
Claims (9)
1. A method for separating and purifying placental blood by using a protective liquid and a placenta extruder is characterized in that the placenta extruder comprises an extrusion rod (1), a fixed rod (2), an extrusion cylinder (3), an extrusion plate (4), a compression-resistant net (5), a filter screen (6) and a fixed seat (8); wherein the extrusion rod (1) is in threaded connection with the fixed rod (2), and the lower end of the extrusion rod (1) is connected with the extrusion plate (4); in the extrusion process, an extrusion disc (4) stretches into the extrusion cylinder (3); a compression-resistant net (5) and a filter screen (6) are arranged below the inside of the extrusion cylinder (3), and the compression-resistant net (5) is arranged above the filter screen (6); the bottom of the extrusion cylinder (3) is conical, and the bottom end of the extrusion cylinder is provided with an extruder sample outlet (7); the fixed seat (8) is fixedly connected to the upper part of the outer wall of the extrusion cylinder (3), and the fixed rod (2) is inserted into the fixed seat (8) and is fixed through a fastening bolt;
the method for separating and purifying the placental blood by using the protective liquid and the placenta extruder comprises the following steps:
1. adding 100 Xdiab into physiological saline as a cleaning solution, wherein the final volume concentration of the 100 Xdiab in the cleaning solution is 1%;
2. weighing sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, L-malic acid and sodium hydroxide, adding into water for injection, stirring to dissolve completely, adjusting pH to 7.0-7.2, and adding low molecular heparin sodium injection and dextran to prepare a protective solution; wherein the final concentration of the low molecular heparin sodium in the protective solution is 25-35IU/mL, the final concentration of the dextran is 0.025g/mL, and the mass ratio of sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium acetate trihydrate, L-malic acid and sodium hydroxide is 3:0.15:0.2:0.1:0.15:0.3:0.1; the mass volume ratio of the sodium chloride to the water for injection is 3g:500mL;
3. washing placenta with washing liquid for 2-3 times, soaking placenta for 4-10min, taking out placenta, washing placenta with protective liquid for 1-2 times, cutting placenta into small pieces, placing on compression-resistant net (5) in placenta extruder, connecting sample outlet (7) of extruder with blood collecting bag, pouring 150-200mL protective liquid into placenta extruder, rotating extrusion rod (1), driving extrusion disc (4), extruding placenta, extruding blood into blood collecting bag, shaking table, mixing for 4-10min, and connecting blood collecting bag with triple bag, wherein the triple bag comprises transfer bag, blood plasma bag and freezing bag; transferring the sample in the blood sampling bag into a transfer bag, sampling and counting, and detecting the total volume of red blood cells in the transfer bag;
if the total volume of the red blood cells in the transfer bag is larger than xmL in the detection result; adding hydroxyethyl starch according to 20% of the total volume of the sample in the transfer bag, uniformly mixing for 4-10min by a shaking table, centrifuging for the first time, discharging red blood cells in the transfer bag into a blood collection bag after centrifuging, discharging the red blood cell volume = the total volume of red blood cells in the transfer bag-xmL, centrifuging for the second time, discharging plasma in the transfer bag into a plasma bag after centrifuging, and discharging the plasma discharge volume = the total volume of the sample in the transfer bag-xmL-discharging the red blood cell volume;
if the total volume of red blood cells in the transfer bag is smaller than xmL in the detection result, directly centrifuging, and discharging the plasma in the transfer bag into a plasma bag after centrifuging, wherein the plasma discharge volume=the total volume of samples in the transfer bag-xmL; wherein xmL is the target volume of cryopreserved placental blood;
4. pre-cooling the transfer bag processed in the step three in a refrigerator, adding frozen stock solution into placenta blood of the transfer bag, fully and uniformly mixing, transferring to the frozen stock bag, taking the frozen stock bag off from the triple bag, sealing, balancing for 4-10min in the refrigerator, transferring to a refrigerator at-20 ℃ for 1-2 hours, transferring to a refrigerator at-80 ℃ for 4-6 hours, and finally transferring to liquid nitrogen at-196 ℃ for freezing; the volume ratio of placenta blood to frozen stock solution in the transfer bag is (5-7): 1.
2. Method according to claim 1, characterized in that the extrusion vessel (3), the extrusion plate (4) and the compression-resistant net (5) are all made of stainless steel.
3. A method according to claim 1, characterized in that the screen (6) is a 100 mesh screen.
4. The method according to claim 1, wherein the 100 Xdiabody is a mixed solution of penicillin and streptomycin.
5. The method of claim 1, wherein the first centrifugation parameters for a volume of red blood cells greater than xmL are: the rotating speed is 50-100g, the time is 5-10min, and the temperature is 10 ℃; the parameters for the second centrifugation were: the rotating speed is 500-800g, the time is 15-20min, and the temperature is 10 ℃; when the volume of the red blood cells is smaller than xmL, the parameters of centrifugation are as follows: the rotating speed is 500-800g, the time is 15-20min, and the temperature is 10 ℃.
6. The method according to claim 1, wherein in step four the transfer bag is pre-cooled in a refrigerator at 4 ℃ for 10min.
7. The method according to claim 1, wherein in the fourth step, the frozen stock solution is prepared from dimethyl sulfone and dextran, the final concentration of the dimethyl sulfone in the frozen stock solution is 50%, and the final concentration of the dextran in the frozen stock solution is 5%.
8. The method according to claim 1, wherein in the fourth step, the freezing bag is placed in a refrigerator at 4 ℃ for balancing for 5min, then transferred into a medical refrigerator at-20 ℃ for 1.5 hours, transferred into a low-temperature refrigerator at-80 ℃ for 5 hours, and finally transferred into liquid nitrogen at-196 ℃ for freezing.
9. A method of resuscitating placental blood cryopreserved according to claim 1, characterized in that the method comprises: balancing frozen placenta blood in a liquid nitrogen tank for 1-3min, transferring to a water bath kettle at 37 ℃ for resuscitation, taking out after thawing, transferring to a centrifuge tube with a protective agent, centrifuging at 1100-1400g and 8-12 ℃ for 5-8min at the volume ratio of the frozen placenta blood to the protective agent of 1 (8-11), and removing the supernatant after centrifuging; wherein the protective agent is a compound electrolyte solution containing human serum albumin and dextran, the final concentration of the human serum albumin in the protective agent is 1%, and the final concentration of the dextran in the protective agent is 5%.
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Family Cites Families (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001017162A (en) * | 1999-07-08 | 2001-01-23 | Asahi Medical Co Ltd | Cell-cleaning method of thawed frozen cell, and cell- cleaning apparatus |
| CN1195055C (en) * | 2001-09-06 | 2005-03-30 | 周胜利 | Method for establishing hematopoietic stem cells bank by extracting hematopoietic cells from placenta tissues |
| US20060182692A1 (en) * | 2003-12-16 | 2006-08-17 | Fishburn C S | Chemically modified small molecules |
| WO2007146105A2 (en) * | 2006-06-05 | 2007-12-21 | Cryo-Cell International, Inc. | Procurement, isolation and cryopreservation of fetal placental cells |
| NL1033365C2 (en) * | 2007-02-09 | 2008-08-12 | Medavinci Dev B V | Device and method for separating and analyzing blood. |
| WO2010043076A1 (en) * | 2008-10-17 | 2010-04-22 | 宁夏医科大学附属医院 | A method for constructing human placental mesenchymal cells library which is suitable for clinical application |
| CN102337245B (en) * | 2011-08-02 | 2012-10-10 | 江苏省北科生物科技有限公司 | Method for separating hemopoietic stem cells through cord blood separation double bag |
| CN105028388A (en) * | 2015-07-08 | 2015-11-11 | 深圳爱生再生医学科技有限公司 | Preserving fluid and preparation method therefor |
| CN105368780B (en) * | 2015-12-14 | 2019-09-24 | 广州赛莱拉干细胞科技股份有限公司 | Lavage fluid, enzymatic hydrolysate and method for separating placenta hematopoietic stem cells |
| CN205443251U (en) * | 2015-12-23 | 2016-08-10 | 天津滨通生物科技有限公司 | Stem cell separation adjustable automatic squeezing mechanism for whole blood bag |
| CN205917261U (en) * | 2016-07-28 | 2017-02-01 | 中国人民解放军第四军医大学 | Primary cells digests quick separation device |
| CN107114356A (en) * | 2016-08-01 | 2017-09-01 | 北京世纪劲得生物技术有限公司 | A kind of placenta and umbilical cord cells protect liquid |
| CN107058224B (en) * | 2017-02-10 | 2020-08-21 | 广东唯泰生物科技有限公司 | Method for extracting and cryopreserving hematopoietic stem cells from placenta |
| WO2018204764A1 (en) * | 2017-05-05 | 2018-11-08 | Camp4 Therapeutics Corporation | Identification and targeted modulation of gene signaling networks |
| CN108148804A (en) * | 2018-01-29 | 2018-06-12 | 浙江喆美生物科技有限公司 | One kind meets clinic bleeding of the umbilicus multipotential stem cell production purification process |
| KR102015502B1 (en) * | 2018-03-29 | 2019-08-28 | 아키소스템바이오스트래티지스(주) | A method for the isolation of stem cells from human umbilical cords |
| CN108812642B (en) * | 2018-07-18 | 2021-07-23 | 银丰生物工程集团有限公司 | Systematic method for preparing placenta tissue according to structural hierarchy and cryopreserving and application |
| CN109090102A (en) * | 2018-09-05 | 2018-12-28 | 成都汇欣生命科技有限公司 | A kind of general serum-free frozen stock solution of mescenchymal stem cell and preparation method thereof |
| CN109481459B (en) * | 2018-11-23 | 2021-07-20 | 济南康和医药科技有限公司 | Compound electrolyte glucose injection and preparation method thereof |
| CN109913416B (en) * | 2019-04-19 | 2022-09-09 | 天晴干细胞股份有限公司 | Preparation method of protective agent used after recovery of frozen umbilical cord blood hematopoietic stem cells |
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