CN110903952A - Method for separating, purifying and recovering placental blood by using protective solution and placental squeezer - Google Patents
Method for separating, purifying and recovering placental blood by using protective solution and placental squeezer Download PDFInfo
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Abstract
A method for separating, purifying and recovering placental blood by using a protective solution and a placental squeezer relates to a method for separating, purifying and recovering placental blood by using a protective solution and a placental squeezer. The invention aims to solve the problems of difficult separation and extraction of placental blood, low rate of activity of nucleated cells, few cells, easy pollution in the preparation process, reduction of cell quality in the recovery process and the like in the conventional method, adopts the placental squeezer and adds a protective solution to fully squeeze out the blood in the placenta, and then ensures that the total cell number of the nucleated cells can reach 15 x 10 by a high-efficiency separation and purification method8Above, the survival rate is above 95%, the clone formation is obvious, and the phenotype flow detection CD34 reaches more than 0.3 percent, the pollution rate is controlled within 1 percent, the survival rate after recovery is more than 90 percent, the cloning capacity is realized, the phenotype flow detection CD34 can reach more than 0.3 percent, and the sterile detection 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, purifying and recovering placental blood by using a protective solution and a placental squeezer.
Background
Placental derived hematopoietic stem cells are a group of primitive hematopoietic cells present in placental tissue, and hematopoietic stem cells are highly undifferentiated cells, which can also be said to be primitive cells of all blood cells (most of which are immune cells).
Transplantation can be used for treating acute leukemia, chronic leukemia, myelodysplastic syndrome, hematopoietic stem cell disease, myeloproliferative disease, lymphoproliferative disease, macrophage disease, hereditary metabolic disease, histiocytic disease, hereditary erythrocyte disease, hereditary immune system disease, hereditary platelet disease, plasma cell disease, thalassemia, non-hematologic malignancy, acute radiation disease, etc. with 75 kinds of lethal diseases such as malignant hematological disease, partial malignancy, partial hereditary disease, etc.
At present, the placental blood is not completely extracted when being stored and separated, a great amount of nucleated cells are lost in the processes of preparation and cryopreservation, and the cell number is 5-10 x 108The preparation time is long, the cells are not sufficiently protected by the normal saline, so that a large number of nucleated cells die, the survival rate is 60-80%, the whole preparation process is basically in an open state, the pollution rate is higher by 15-20%, and the quality is reduced more obviously after freezing recovery.
Disclosure of Invention
The invention aims to solve the problems of difficult separation and extraction of placental blood, low survival rate of nucleated cells, few cells, long preparation time, low phenotypic detection value and easy pollution in the preparation process in the existing method, and provides a method for separating, purifying and recovering placental blood by using a protective solution and a placental squeezer.
The invention relates to a placenta squeezer which comprises a squeezing rod, a fixing rod, a squeezing barrel, a squeezing disc, a compression-resistant net, a filter screen and a fixing seat; wherein 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 extends into the extrusion cylinder; a compression-resistant net and a filter screen are arranged below the interior of the extrusion cylinder, and the compression-resistant net is arranged above the filter screen; the bottom of the extruding cylinder is conical, and the bottom end of the extruding cylinder is provided with a pipeline connecting port; the fixing seat is fixedly connected to the upper portion of the outer wall of the extrusion barrel, and the fixing rod is inserted into the fixing seat and fixed through the fastening 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 drawing the fixing rod, so that the placenta extruder is conveniently placed in the placenta or impurities such as blood coagulation and tissues in hematopoiesis are conveniently removed. When the placenta blood collecting device is used, the cleaned placenta tissue blocks are placed on the compression-resistant net in the extruding cylinder, the extruding rod is rotated to drive the extruding disc to extrude placenta tissues, blood in the placenta is filtered by the filter screen and is discharged into the blood collecting bag through the pipeline connecting port, 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 solution and a placental squeezer, which comprises the following steps:
firstly, 100 Xdouble antibody is added into physiological saline to be used as cleaning solution, and the final volume concentration of the 100 Xdouble antibody in the cleaning solution is 1 percent;
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 until the sodium chloride, the potassium chloride, the calcium chloride dihydrate, the magnesium chloride hexahydrate, the sodium acetate trihydrate, the L-malic acid and the sodium hydroxide are completely dissolved, adjusting the pH value to 7.0-7.2, adding low molecular weight heparin sodium injection and dextran, and preparing into 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 the sodium chloride, the potassium chloride, the calcium chloride dihydrate, the magnesium chloride hexahydrate, the sodium acetate trihydrate, the L-malic acid and the 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;
washing the placenta for 2-3 times by using a cleaning solution, soaking the placenta for 4-10min by using the cleaning solution, taking out the placenta, cleaning the placenta for 1-2 times by using a protective solution, shearing the placenta into small blocks, placing the small blocks on a compression-resistant net in a placenta squeezer, connecting a sample outlet of the squeezer with a blood collection bag, pouring 200mL of the protective solution into the placenta squeezer, rotating a squeezing rod to drive a squeezing disc to squeeze the placenta, squeezing blood into the blood collection bag, uniformly mixing the blood in a shaking table for 4-10min, and connecting the blood collection bag with a triple bag, wherein the triple bag is divided into a transfer bag, a plasma bag and a cryopreservation bag; transferring the sample in the blood collection bag into a transfer bag, sampling and counting, and detecting the total volume of the red blood cells in the transfer bag;
if the total volume of the red blood cells in the transfer bag is more 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 using a shaker, then carrying out primary centrifugation, discharging red blood cells in the transfer bag into a blood collection bag after centrifugation, wherein the volume of the discharged red blood cells is equal to the total volume-x mL of the red blood cells in the transfer bag, then carrying out secondary centrifugation, discharging plasma in the transfer bag into a plasma bag after centrifugation, and the volume of the discharged plasma is equal to the total volume-x mL-of the sample in the transfer bag;
if the total volume of the red blood cells in the transfer bag is less than xmL in the detection result, directly performing centrifugation, discharging the plasma in the transfer bag into a plasma bag after the centrifugation, wherein the discharged volume of the plasma is-x mL of the total volume of the sample in the transfer bag; wherein x mL is the target volume of cryopreserved placental blood;
fourthly, placing the transfer bag processed in the third step into a refrigerator for precooling, then adding the frozen stock solution into the placental blood of the transfer bag, fully and uniformly mixing, transferring the mixture into the frozen stock bag, taking the frozen stock bag off the triple bag, sealing, then placing the triple bag into the refrigerator for balancing for 4-10min, then transferring the triple bag into the refrigerator at-20 ℃ for 1-2 h, then transferring the triple bag into the refrigerator at-80 ℃ for 4-6 h, and finally transferring the triple bag into liquid nitrogen at-196 ℃ for freezing, thus completing the process; the volume ratio of the placental blood to the frozen stock solution in the transfer bag is (5-7) to 1.
The method for recovering the placental blood comprises the following steps: placing the frozen placental blood in a liquid nitrogen tank for balancing for 1-3min, then transferring to a 37 ℃ water bath pot for resuscitation, taking out after thawing, transferring to a centrifuge tube with a protective agent, wherein the volume ratio of the frozen placental blood to the protective agent is 1 (8-11), centrifuging for 5-8min at the rotation speed of 1100-1400g and at the temperature of 8-12 ℃, and removing the supernatant after the centrifugation is finished, thus completing the preparation; the protective agent is a compound electrolyte solution containing human serum albumin and dextran, the volume final concentration of the human serum albumin in the protective agent is 1%, and the volume final concentration of the dextran is 5%.
The invention uses self-prepared protective liquid, 3 to 4g of sodium chloride, 0.15 to 0.2g of potassium chloride, 0.2 to 0.3g of calcium chloride dihydrate, 0.1 to 0.2g of magnesium chloride hexahydrate, 0.15 to 0.2g of sodium acetate trihydrate, 0.3 to 0.4g of L-malic acid and 0.1 to 0.2g of sodium hydroxide are weighed, added into 530mL of water for injection of 490-90 mL and stirred until completely dissolved, the pH value is adjusted to be between 7.0 and 7.2, then low molecular heparin sodium injection and dextran are added, the concentration of the low molecular heparin sodium is 25 to 35/mL, the final concentration of the dextran is 0.025g/mL, the protective liquid is prepared, the survival rate, the non-agglomeration and the outstanding cloning capacity of nucleated cells of a sample can be ensured in the preparation and cryopreservation processes, and the phenotype CD34 is detected to be more than 0.3 percent.
The invention has the beneficial effects that:
the invention adopts the placenta squeezer to fully squeeze out the blood in the placenta, the protective solution plays a certain role in protecting the nucleated cells, and then the high-efficiency separation and purification method ensures that the total cell number of the nucleated cells in the prepared placenta blood can reach 15 x 108The survival rate is more than 95 percent, the clone formation is obvious, the phenotype flow detection CD34 reaches more than 0.3 percent, the pollution rate is controlled within 1 percent, the preparation time is controlled within 2.5 hours, the frozen stock solution is added to be cooled to minus 80 ℃, the frozen stock solution is transferred into liquid nitrogen to be stored for 6 months and then recovered, the survival rate is more than 90 percent after the recovery by using a new recovery method, the clone capability is realized, the phenotype flow detection CD34 can reach more than 0.3 percent, and the aseptic detection is negative.
Drawings
Fig. 1 is a schematic structural view of the placenta squeezer of the present invention;
fig. 2 is a schematic view of a compression-resistant net and a filter net of the placenta squeezer;
FIG. 3 is a graph showing the differentiation of colonies isolated and purified in example 1 before freezing of nucleated cells in placental blood;
FIG. 4 is a graph showing the differentiation of colonies recovered after freezing the nucleated cells in the placental blood isolated and purified in example 1.
Detailed Description
The first embodiment is as follows: the placenta extruder comprises an extrusion rod 1, a fixed rod 2, an extrusion cylinder 3, an extrusion disc 4, a pressure-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 disc 4; in the extrusion process, the extrusion disc 4 extends into the extrusion cylinder 3; a pressure-resistant net 5 and a filter screen 6 are arranged below the interior of the extrusion container 3, and the pressure-resistant net 5 is arranged above the filter screen 6; the bottom of the extrusion container 3 is conical, and the bottom end is provided with a pipeline connecting port 7; fixing base 8 fixed connection is on the upper portion of 3 outer walls of recipient, and dead lever 2 is pegged graft on fixing base 8, and is fixed through holding bolt.
In this embodiment, the diameter of the extrusion disk 4, the diameter of the compression net 5 and the diameter of the filter net 6 are equal.
The fixing base 8 of the placenta extruder of the embodiment is provided with the fastening bolt, the fixing rod 2 which is fixedly connected with the fixed bolt in an inserting mode is used for unscrewing the bolt, the extruding disc 4 can be pulled out from the extruding cylinder 3 through the drawing fixing rod 2, and the placenta is conveniently placed in or impurities such as blood coagulation and tissues in hematopoiesis are conveniently removed. When the placenta collecting device is used, the cleaned placenta tissue blocks are placed on the compression-resistant net 5 in the extruding cylinder 3, the extruding rod 1 is rotated to drive the extruding disc 4 to extrude placenta tissues, blood in the placenta is filtered by the filter screen 6 and is discharged into a blood collecting bag through the pipeline connecting port 7.
The placenta squeezer and the protective solution of the embodiment are adopted to fully squeeze out 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 nucleated cells in the placenta blood can reach 15 x 108The survival rate is more than 95 percent, the clone formation is obvious, the phenotype flow detection CD34 reaches more than 0.3 percent, the pollution rate is controlled within 1 percent, frozen stock solution is added for gradient cooling to 80 ℃ below zero, the frozen stock solution is transferred into liquid nitrogen for storage for 6 months and then revived, the survival rate is more than 90 percent after reviving, the clone capability is realized, the phenotype flow detection CD34 can reach more than 0.3 percent, and the sterile detection is negative.
The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: the extrusion cylinder 3, the extrusion disc 4 and the pressure resistant net 5 are all made of stainless steel. The rest is the same as the first embodiment.
The third concrete implementation mode: the present embodiment differs from the first or second embodiment in that: the filter screen 6 is a 100-mesh filter screen. The others are the same as in the first or second embodiment.
The fourth concrete implementation mode: the method for separating and purifying the placental blood by using the protective solution and the placental squeezing device is carried out according to the following steps:
firstly, 100 Xdouble antibody is added into physiological saline to be used as cleaning solution, and the final volume concentration of the 100 Xdouble antibody in the cleaning solution is 1 percent;
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 until the sodium chloride, the potassium chloride, the calcium chloride dihydrate, the magnesium chloride hexahydrate, the sodium acetate trihydrate, the L-malic acid and the sodium hydroxide are completely dissolved, adjusting the pH value to 7.0-7.2, adding low molecular weight heparin sodium injection and dextran, and preparing into 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 the sodium chloride, the potassium chloride, the calcium chloride dihydrate, the magnesium chloride hexahydrate, the sodium acetate trihydrate, the L-malic acid and the 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;
washing the placenta for 2-3 times by using a cleaning solution, soaking the placenta for 4-10min by using the cleaning solution, taking out the placenta, cleaning the placenta for 1-2 times by using a protective solution, cutting the placenta into small blocks, placing the small blocks on a pressure resistant net 5 in a placenta squeezer, connecting a sample outlet 7 of the squeezer with a blood collection bag, pouring 150 plus 200mL of protective solution into the placenta squeezer, rotating a squeezing rod 1 to drive a squeezing disc 4 to squeeze the placenta, squeezing blood into the blood collection bag, uniformly mixing the blood with a shaking table for 4-10min, and connecting the blood collection bag with a triple bag, wherein the triple bag is divided into a transfer bag, a plasma bag and a cryopreservation bag; transferring the sample in the blood collection bag into a transfer bag, sampling and counting, and detecting the total volume of the red blood cells in the transfer bag;
if the total volume of the red blood cells in the transfer bag is more 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 using a shaker, then carrying out primary centrifugation, discharging red blood cells in the transfer bag into a blood collection bag after centrifugation, wherein the volume of the discharged red blood cells is equal to the total volume-x mL of the red blood cells in the transfer bag, then carrying out secondary centrifugation, discharging plasma in the transfer bag into a plasma bag after centrifugation, and the volume of the discharged plasma is equal to the total volume-x mL-of the sample in the transfer bag;
if the total volume of the red blood cells in the transfer bag is less than xmL in the detection result, directly performing centrifugation, discharging the plasma in the transfer bag into a plasma bag after the centrifugation, wherein the discharged volume of the plasma is-x mL of the total volume of the sample in the transfer bag; wherein x mL is the target volume of cryopreserved placental blood;
fourthly, placing the transfer bag processed in the third step into a refrigerator for precooling, then adding the frozen stock solution into the placental blood of the transfer bag, fully and uniformly mixing, transferring the mixture into the frozen stock bag, taking the frozen stock bag off the triple bag, sealing, then placing the triple bag into the refrigerator for balancing for 4-10min, then transferring the triple bag into the refrigerator at-20 ℃ for 1-2 h, then transferring the triple bag into the refrigerator at-80 ℃ for 4-6 h, and finally transferring the triple bag into liquid nitrogen at-196 ℃ for freezing, thus completing the process; the volume ratio of the placental blood to the frozen stock solution in the transfer bag is (5-7) to 1.
The fifth concrete implementation mode: the fourth difference between this embodiment and the specific embodiment is that: the 100 Xdouble antibody is a mixed solution of penicillin and streptomycin. The rest is the same as the fourth embodiment.
The sixth specific implementation mode: the fourth or fifth embodiment is different from the specific embodiment in that: the first centrifugation parameters when the volume of erythrocytes was greater than xmL were: rotating at 50-100g for 5-10min at 10 deg.C; the parameters of the second centrifugation were: the rotating speed is 500-800g, the time is 15-20min, and the temperature is 10 ℃; when the volume of red blood cells is less than xmL, the parameters of centrifugation are: the rotation speed is 500-. The others are the same as the fourth or fifth embodiments.
The seventh embodiment: this embodiment differs from one of the fourth to sixth embodiments in that: and step four, placing the transfer bag into a refrigerator with the temperature of 4 ℃ for precooling for 10 min. The rest is the same as one of the fourth to sixth embodiments.
The specific implementation mode is eight: this embodiment is different from one of the fourth to seventh embodiments in that: the frozen stock solution in the fourth step is prepared from dimethyl sulfone and dextran, the volume final concentration of the dimethyl sulfone in the frozen stock solution is 50%, and the volume final concentration of the dextran is 5%. The rest is the same as one of the fourth to seventh embodiments.
The specific implementation method nine: this embodiment is different from the fourth to eighth embodiment in that: in the fourth step, the freezing bag is put into a refrigerator with the temperature of 4 ℃ for 5min for balancing, then is transferred into a medical refrigerator with the temperature of 20 ℃ below zero for 1.5 hours, then is transferred into a refrigerator with the temperature of 80 ℃ below zero for 5 hours, and finally is transferred into liquid nitrogen with the temperature of 196 ℃ below zero for freezing. The rest is the same as the fourth to eighth embodiments.
The detailed implementation mode is ten: the method for recovering the placental blood in the embodiment comprises the following steps: placing the frozen placental blood in a liquid nitrogen tank for balancing for 1-3min, then transferring to a 37 ℃ water bath pot for resuscitation, taking out after thawing, transferring to a centrifuge tube with a protective agent, wherein the volume ratio of the frozen placental blood to the protective agent is 1 (8-11), centrifuging for 5-8min at the rotation speed of 1100-1400g and at the temperature of 8-12 ℃, and removing the supernatant after the centrifugation is finished, thus completing the preparation; the protective agent is a compound electrolyte solution containing human serum albumin and dextran, the volume final concentration of the human serum albumin in the protective agent is 1%, and the volume final concentration of the dextran is 5%.
The effect of the invention is demonstrated by the following examples:
example 1: a method for separating and purifying placental blood by using a protective solution and a placental squeezer comprises the following steps:
firstly, adding 5mL of 100 Xdouble antibody (penicillin and streptomycin) into 500mL of physiological saline to obtain a cleaning solution;
secondly, preparing a protective solution in advance, weighing 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, adding the above materials into 500ml of water for injection, stirring until the materials are completely dissolved, adjusting the pH value to 7.0-7.2 by using hydrochloric acid or sodium hydroxide, and adding a low-molecular heparin sodium injection and dextran 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 0.025 g/mL.
Taking out the sterilized tray, hemostatic forceps and tissue scissors from the instrument box, clamping the placenta from a sterile bag by the hemostatic forceps, putting the placenta into the tray, removing umbilical cord, amnion, blood coagulation and grease impurities on the placenta by the tissue scissors, washing the placenta for 3 times by using a cleaning solution, soaking the placenta for 5min by using the cleaning solution, taking out the placenta, cleaning the placenta for 2 times by using a protective solution, cutting the placenta into small blocks, putting the small blocks on a pressure resistant net 5 in a placenta squeezer, connecting a sample outlet 7 of the squeezer with a blood collection bag, pouring 150mL of the protective solution into the placenta squeezer, rotating a squeezing rod 1 to drive a squeezing disc 4, squeezing the placenta, squeezing the blood into the blood collection bag, uniformly mixing the blood for 5min by a shaking table, and connecting the blood collection bag with a triple bag, wherein the triple bag is divided into a transfer bag, a blood plasma bag and a cryopreservation bag; transferring the sample in the blood collection 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 more 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 the mixture by using a shaking table for 4-10min, then carrying out first centrifugation (50-100g, 5-10min and 10 ℃), placing the mouth of the transfer bag downwards during centrifugation, placing red blood cells at the mouth position after centrifugation, opening the pipe clamps of the three-link bag, discharging the red blood cells in the transfer bag into a blood collection bag, discharging the volume of the red blood cells to be equal to-20 mL of the total volume of the red blood cells in the transfer bag, then carrying out second centrifugation, placing the mouth of the transfer bag upwards during centrifugation, opening the pipe clamps of the three-link bag after centrifugation, discharging the plasma in the transfer bag into a plasma bag in an extruding manner, and discharging the volume of the plasma to be equal to-20 mL of the total volume of the sample in the transfer bag;
if the total volume of the red blood cells in the transfer bag is less than 20mL in the detection result, directly performing centrifugation (800 g, 15-20min and 10 ℃), discharging the plasma in the transfer bag into the plasma bag after the centrifugation, wherein the discharged volume of the plasma is-20 mL of the total volume of the sample in the transfer bag; and (4) fully and uniformly mixing the residual samples in the transfer bag, and sampling for inspection.
The conventional method of the comparative group is as follows: cleaning with normal saline as a protective agent, adding nothing for protection, cutting into blocks after cleaning, transferring into a sterile bag, extruding blood into a tray by hand extrusion, pouring normal saline into the sterile bag, repeatedly cleaning and extruding twice, filtering liquid in the tray by a 100-mesh stainless steel funnel, transferring into a 50ml centrifuge tube for centrifugation, recovering upper plasma, injecting into a sterile detection bottle, recovering a middle white film layer into a new centrifuge tube, fully mixing, sampling and inspecting.
For the comparison group of 15 batches of the conventional preparation method and the new preparation method, samples were taken for nucleated cell counting, nucleated cell viability, flow-type CD34 phenotype detection, clone colony formation, sterility and preparation time statistics, and the results are shown in Table 1.
Fourthly, placing the transfer bag processed in the third step into a refrigerator for precooling, then adding the frozen stock solution into the transfer bag, fully and uniformly mixing, placing the transfer bag into a refrigerator with the temperature of 4 ℃ for balancing for 5min, then transferring the transfer bag into a medical refrigerator with the temperature of 20 ℃ below zero for 1.5 hours, then transferring the transfer bag into a refrigerator with the temperature of 80 ℃ below zero for 5 hours, and finally transferring the transfer bag into liquid nitrogen with the temperature of 196 ℃ below zero for freezing, thus completing the process; the volume ratio of the placental blood to the frozen stock solution in the transfer bag is 6: 1.
The method for recovering the cryopreserved placental blood comprises the following steps: and (3) balancing the sample in a tank opening of a liquid nitrogen tank for 1min, then quickly transferring to a water bath kettle at 37 ℃ for resuscitation, taking out the sample in time after the sample is melted, transferring the sample into a 50ml centrifugal tube with a protective agent, wherein the ratio of the sample to the protective solution is 1: 10. The protective agent is a compound electrolyte solution containing human serum albumin and dextran, the final volume concentration of the human serum albumin in the protective agent is 1%, the final volume concentration of the dextran is 5%, the centrifugation is carried out at 1300 rpm, 7 minutes and 10 ℃, the supernatant is extracted and injected into a sterile detection bottle after the centrifugation is finished, the rest part is discarded, and the bottom sample is sent for detection of the survival rate, the phenotype, the formation of clone colonies and the counting; wherein the supernatant is a protective agent.
The conventional method of the comparative group is as follows: adjusting the temperature of the water bath to 37 ℃ in advance, taking out the frozen placental blood from the liquid nitrogen, putting the placental blood into the water bath for 2min, then transferring the placental blood into physiological saline, centrifuging (1300-1500 rpm, 5-10min and 10 ℃), extracting the supernatant, injecting the supernatant into a detection bottle for detecting sterility, and sending the rest samples to be detected for nucleated cell number, viability, clonal colony formation and flow-type CD34 phenotypic detection.
Taking 2 tubes of 15 batches of frozen samples, respectively carrying out recovery detection by using a conventional recovery method and a new recovery method, and comparing the number of nucleated cells, the survival rate, the formation of clone colonies, the phenotypic detection of flow-type CD34 and the sterile detection. The comparative results 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 disc 4, a pressure-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 disc 4; in the extrusion process, the extrusion disc 4 extends into the extrusion cylinder 3; a pressure-resistant net 5 and a filter screen 6 are arranged below the interior of the extrusion container 3, and the pressure-resistant net 5 is arranged above the filter screen 6; the bottom of the extrusion container 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 barrel 3, and the fixed rod 2 is inserted into the fixed seat 8 and fixed through a fastening bolt; wherein a schematic view of the compression net 5 and the sieve 6 is shown in figure 2.
TABLE 1
From table 1, it can be seen that the number of nucleated cells, the survival rate, the expression of phenotype CD34, the colony formation, and the average value of the purified placental blood obtained by using the placental squeezer and the protective solution are all much higher than those of the conventional preparation method, and the preparation time of the novel method is also reduced, the average value of the bacterial infection and positive report rate of the novel sterile detection method of this batch is 0, and the average value of the conventional method is 15.9. The new preparation method can improve the quality of the sample.
TABLE 2
From table 2, it can be seen that the conventional resuscitation method is greatly reduced in survival rate, cell number, detection of flow-type phenotype CD34, and colony formation compared with the sample before cryopreservation, and the novel resuscitation method is reduced in reduction and resuscitation compared with the sample before cryopreservation, so that the novel resuscitation method plays a certain role in sample protection.
As can be seen from table 1, the collection amount of nucleated cells in the placental blood is greatly increased by using the squeezer of the present embodiment to collect the placental blood, the preparation time is reduced, the work efficiency is ensured, the placental blood is protected by the protection solution, the osmotic pressure of placental blood cells is ensured, cell agglomeration and apoptosis are avoided, the survival rate is relatively increased, the formation of clonal colony and the expression of flow CD34 are relatively increased, and the contamination rate is controlled to be less than 1%, as can be seen from table 2, the cells in the same batch are compared with the new recovery method by using the conventional recovery method, through the action of the protective agent in the cryopreservation frozen storage solution and the recovery process, the new method is superior to the conventional recovery method in the detection quality of the nucleated cell survival rate, the nucleated cell number, the clonality detection, the expression of flow CD34 and the like of the sample in the same batch as that in the new batch, as can be compared with the clonality of the new recovery method by using the preparation method in fig. 3 and fig. 4, the method has no obvious difference and strong cloning capacity, and ensures that the sample pollution rate is controlled below 1% by cleaning the cleaning solution at the early stage and separating by using a triple bag in the whole process; the triple bag of this example was purchased from Thermogenesis.
Claims (10)
1. A placenta squeezer is characterized by comprising a squeezing rod (1), a fixing rod (2), a squeezing barrel (3), a squeezing disc (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 disc (4); in the extrusion process, the extrusion disc (4) extends into the extrusion cylinder (3); a pressure-resistant net (5) and a filter screen (6) are arranged below the interior of the extrusion cylinder (3), and the pressure-resistant net (5) is arranged above the filter screen (6); the bottom of the extrusion container (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 portion of the outer wall of the extrusion container (3), and the fixing rod (2) is inserted into the fixing seat (8) and fixed through the fastening bolt.
2. Placenta press according to claim 1, characterized in that the pressing cylinder (3), the pressing plate (4) and the pressure-resistant mesh (5) are made of stainless steel.
3. The placental squeezer according to claim 1, wherein the sieve (6) is a 100 mesh sieve.
4. A method for separating and purifying placental blood by using a protective solution and the placental extractor of claim 1, the method comprising the steps of:
firstly, 100 Xdouble antibody is added into physiological saline to be used as cleaning solution, and the final volume concentration of the 100 Xdouble antibody in the cleaning solution is 1 percent;
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 until the sodium chloride, the potassium chloride, the calcium chloride dihydrate, the magnesium chloride hexahydrate, the sodium acetate trihydrate, the L-malic acid and the sodium hydroxide are completely dissolved, adjusting the pH value to 7.0-7.2, adding low molecular weight heparin sodium injection and dextran, and preparing into 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 the sodium chloride, the potassium chloride, the calcium chloride dihydrate, the magnesium chloride hexahydrate, the sodium acetate trihydrate, the L-malic acid and the 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;
washing the placenta for 2-3 times by using a cleaning solution, soaking the placenta for 4-10min by using the cleaning solution, taking out the placenta, cleaning the placenta for 1-2 times by using a protective solution, shearing the placenta into small blocks, placing the small blocks on a pressure resistant net (5) in a placenta squeezer, connecting a sample outlet (7) of the squeezer with a blood collection bag, pouring 150 plus 200mL of the protective solution into the placenta squeezer, rotating a squeezing rod (1), driving a squeezing disc (4) to squeeze the placenta, squeezing the blood into the blood collection bag, uniformly mixing a shaking table for 4-10min, and connecting the blood collection bag with a triple bag, wherein the triple bag is divided into a transfer bag, a plasma bag and a cryopreservation bag; transferring the sample in the blood collection bag into a transfer bag, sampling and counting, and detecting the total volume of the red blood cells in the transfer bag;
if the total volume of the red blood cells in the transfer bag is more 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 using a shaker, then carrying out primary centrifugation, discharging red blood cells in the transfer bag into a blood collection bag after centrifugation, wherein the volume of the discharged red blood cells is equal to the total volume-x mL of the red blood cells in the transfer bag, then carrying out secondary centrifugation, discharging plasma in the transfer bag into a plasma bag after centrifugation, and the volume of the discharged plasma is equal to the total volume-x mL-of the sample in the transfer bag;
if the total volume of the red blood cells in the transfer bag is less than xmL in the detection result, directly performing centrifugation, discharging the plasma in the transfer bag into a plasma bag after the centrifugation, wherein the discharged volume of the plasma is-x mL of the total volume of the sample in the transfer bag; wherein x mL is the target volume of cryopreserved placental blood;
fourthly, placing the transfer bag processed in the third step into a refrigerator for precooling, then adding the frozen stock solution into the placental blood of the transfer bag, fully and uniformly mixing, transferring the mixture into the frozen stock bag, taking the frozen stock bag off the triple bag, sealing, then placing the triple bag into the refrigerator for balancing for 4-10min, then transferring the triple bag into the refrigerator at-20 ℃ for 1-2 h, then transferring the triple bag into the refrigerator at-80 ℃ for 4-6 h, and finally transferring the triple bag into liquid nitrogen at-196 ℃ for freezing, thus completing the process; the volume ratio of the placental blood to the frozen stock solution in the transfer bag is (5-7) to 1.
5. The method for separating and purifying placental blood according to claim 4, wherein the 100 x diabody is a mixed solution of penicillin and streptomycin.
6. The method for separating and purifying placental blood using a protective solution and a placental squeezer, according to claim 4, wherein the first centrifugation parameter for a volume of red blood cells greater than x mL is: rotating at 50-100g for 5-10min at 10 deg.C; the parameters of the second centrifugation were: the rotating speed is 500-800g, the time is 15-20min, and the temperature is 10 ℃; when the volume of red blood cells is less than xmL, the parameters of centrifugation are: the rotation speed is 500-.
7. The method for separating and purifying placental blood according to claim 4, wherein the step four comprises pre-cooling the transfer bag in a refrigerator at 4 ℃ for 10 min.
8. The method of claim 4, wherein the freezing medium is made of dimethyl sulfone and dextran, the freezing medium has a final volume concentration of dimethyl sulfone of 50% and the dextran has a final volume concentration of 5%.
9. The method for separating and purifying placental blood according to claim 4, wherein the cryopreserved bag is placed in a refrigerator at 4 ℃ for 5min, then transferred to a medical refrigerator at-20 ℃ for 1.5 hours, then transferred to a low-temperature refrigerator at-80 ℃ for 5 hours, and finally transferred to liquid nitrogen at-196 ℃ for cryopreservation.
10. A method of resuscitating cryopreserved placental blood of claim 4, characterized in that the method comprises: placing the frozen placental blood in a liquid nitrogen tank for balancing for 1-3min, then transferring to a 37 ℃ water bath pot for resuscitation, taking out after thawing, transferring to a centrifuge tube with a protective agent, wherein the volume ratio of the frozen placental blood to the protective agent is 1 (8-11), centrifuging for 5-8min at the rotation speed of 1100-1400g and at the temperature of 8-12 ℃, and removing the supernatant after the centrifugation is finished, thus completing the preparation; the protective agent is a compound electrolyte solution containing human serum albumin and dextran, the volume final concentration of the human serum albumin in the protective agent is 1%, and the volume final concentration of the dextran is 5%.
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