CN111254113A - Method for extracting placenta hematopoietic stem cells - Google Patents

Abstract

The invention discloses a method for extracting placenta hematopoietic stem cells, which comprises the following steps: cleaning placenta, sterilizing, detecting maternal blood, and extracting hematopoietic stem cells. The preparation method has the advantages of simplified preparation process, convenient operation and reduced mechanical damage; the blood does not leave the sterile environment in the whole process, and the contamination rate is reduced.

Description

Method for extracting placenta hematopoietic stem cells

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of biology, in particular to a method for extracting placenta hematopoietic stem cells.

[ background of the invention ]

The human placenta is a ligament for conveying various nutrient substances for the survival of babies, and consists of an amnion, a chorion and a decidua basalis. The amnion and chorion originate from the fetus, and the decidua basalis in different regions originate from the mother. The placenta, in addition to playing an important role in fetal development, nutritional support and maintenance of tolerance, is a reservoir of stem cells. Since placenta-derived hematopoietic stem cells can be differentiated into various blood cells and immune cells, they can be used for the treatment of malignant blood diseases in the future as in umbilical cord blood. The study of the human placenta found that the placenta is a hematopoietic organ. From the third week of pregnancy to birth, the placenta also plays a role in hematopoietic function while performing the functions of substance exchange, immune regulation, secretion of active factors, and the like.

The hemopoietic stem cell extracted from placenta has the advantages of rich content, low rejection reaction after transplantation, no ethical barrier and the like, and also has the advantages of earlier hemopoietic stem cell in placenta and strong proliferation capacity; the hematopoietic stem cells of placenta can also be used for preparing red blood cells and megakaryocytes/platelets in large scale and replacing blood transfusion.

In view of the advantages of the placenta hematopoietic stem cells, the development of the placenta tissue hematopoietic stem cell bank technology is a great progress in the stem cell research field, the technical problems of insufficient sources of bone marrow or mobilized peripheral blood, insufficient amount of umbilical cord blood and the like during transplantation can be effectively solved, and the placenta hematopoietic stem cells are expected to replace the bone marrow, the mobilized peripheral blood and the umbilical cord blood to be used for treating malignant blood system diseases by hematopoietic stem cell transplantation, so that the extraction of the placenta hematopoietic stem cells is of great importance.

The most recent technical scheme currently available is as follows: a method for obtaining hematopoietic stem cells by cutting placenta tissue into pieces and washing, but the obtained stem cells are small in number and easy to contaminate. Therefore, we propose a method for placental hematopoietic stem cell extraction.

[ summary of the invention ]

The invention mainly aims to provide a method for extracting placenta hematopoietic stem cells, which can effectively solve the problems in the background technology.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for extracting placenta hematopoietic stem cells comprises the following steps:

the method comprises the following steps: the placenta was transported to a laboratory, EP tubes, 5 of which were labeled with maternal blood "m" and 4 of which were labeled with cord blood "Q", were prepared and frozen bags were pasted with bar codes. Taking 1ml and 0.5ml of cord blood whole blood into 2 EP tubes (1ml is frozen and stored, 0.5ml is used for blood type detection), then centrifuging the residual cord blood and maternal blood at 1500rpm/5min and subpackaging, centrifuging the cord blood and subpackaging supernatant, filling the supernatant into 2 EP tubes, wherein 0.5ml of each EP tube is frozen and stored, centrifuging the maternal blood and subpackaging the supernatant into 4 EP tubes, wherein 3 EP tubes are frozen and stored for rechecking and rechecking, about 0.5ml of each EP tube is used, and 1 EP tube is sent for detection after being subpackaged according to the detection dosage of a single time by a third party and is used for five infectious diseases and ALT detection; the mother blood cells were transferred to 1 EP tube, frozen and stored together with the mother plasma and cord blood, and the sample storage site was registered.

Step two: washing placenta with NA until the color of the washing solution is light red, and washing placenta at least 15 times.

Step three: the umbilical cord in the placenta is connected with one end of a pump through a pump pipe, the other end of the pump is connected with one bag in the triple bag through the pump pipe, the three bags are connected with each other through connecting pipes, the pump is turned on, fetal blood distributed in the placental villus microvessels is pumped out, and the fetal blood is moved to the triple bag.

Step four: connecting the villus microvessels in the placenta with one end of a catheter with a needle, placing the other end of the catheter with the needle in digestive juice, starting a pump, and completely cleaning the residual blood in the placenta.

Step five: after the cleaning, the pump is closed, and the triple bag blood sampling pipeline is thermally sealed.

Step six: adopting a pressing pulp method to extract the hematopoietic stem cells.

Step seven: the blood bag was mixed on a shaker for 5min and a small portion was shunted into a small transfer bag.

Step eight: placing the uniformly mixed blood bag on a double-face super clean bench, sterilizing the blood bag by using an iodophor cotton ball and an ethanol disinfectant cotton ball, extracting 2.5ml of HSC sample blood from the small transfer bag, subpackaging the blood sample into 3 bar-code-pasted EP tubes, wherein the volume of the HSC sample blood sample is 1.5ml, 0.5ml and 0.5ml respectively (1.5 ml is used for ex-warehouse HSC rechecking and is reserved at the temperature of minus 20 ℃, 0.5ml is subpackaged again and is respectively used for cell counting, activity determination and blood type determination, and 0.5ml is used for CFU-GM culture), and if typing is carried out, adding 1.5ml of sample for HLA typing detection.

Step nine: adding HES according to blood volume, sterilizing with iodophor and ethanol disinfectant cotton ball in double-side superclean bench, adding HES into blood bag, and mixing in shaking table for 5 min.

Step ten: the cell number was analyzed by a hemograph.

Step eleven: cell activity assay

Step twelve: the blood bag is put into a large-scale floor type refrigerated centrifuge after being balanced, the temperature is 4-10 ℃, the rotating speed is 480rpm, and the centrifugation is carried out for 6 min.

Step thirteen: and (6) grouting.

Fourteen steps: and (3) putting the blood bag back to the original position in the large floor type refrigerated centrifuge, centrifuging for 5min at the temperature of 4-10 ℃ and the rotating speed of 1500 rpm.

Step fifteen: and (6) grouting.

Sixthly, the steps are as follows: repeating the steps 15-16.

Seventeen steps: after high-speed centrifugation, separating partial plasma in the transfer bag into a sample collection bag (controlling 20-40 ml of blood in the transfer bag) by using a plasma separation clamp, keeping the plasma in a blood bag conduit, after heat-sealing and shearing off one end of the transfer bag, extracting the plasma in the conduit by using an injector in a clean A-level area, respectively injecting the plasma into 3 EP tubes, storing the plasma together with HSC sample whole blood, maternal blood plasma and maternal blood cells in a refrigerator (8 tubes in total) at the temperature of-20 ℃ for rechecking, and closing the clamp on the blood bag conduit to heat-seal and shear off the partial conduit.

Eighteen steps: and taking the heat-sealed sample collecting bag for inoculating the bacteria detection bottle for bacterial culture.

Nineteen steps: mixing the transferring bags containing leucocyte, sterilizing (iodophor and ethanol disinfectant) outer port of blood bag in A-grade workbench, taking out concentrated leucocyte with 50ml syringe, transferring into freezing bag, and collecting blood sample 0.5ml for cell classification and counting and CD34+ CD45dim flow cytometry after preparation.

Twenty steps: preparing a cryoprotectant, extracting a proper amount of DMSO and dextran according to the volume of the HSC in the freezing bag and a conversion table to ensure that the final concentration of the DMSO protected cells is 9.8%, uniformly mixing, placing in a refrigerator with the temperature of-20 ℃ for cooling and balancing for 5-15min to below 4 ℃, and simultaneously placing the HSC freezing bag in the refrigerator with the temperature of 4 ℃ for balancing for 15 min.

Twenty one: connecting the tube mouth of the disinfection freezing bag with a corresponding injector according to the bar code number, flatly placing the freezing bag on a horizontal shaking table for fixing, fixing the injector on a 10-channel automatic injection pump, slowly and uniformly injecting the cell freezing protective agent into the freezing bag, and discharging the residual cell freezing protective agent in the injector and the catheter into the HSC, so that the HSC and the cell freezing protective agent are fully mixed, and the cell freezing injury phenomenon is avoided.

Step twenty-two: and (3) heat-sealing off a catheter of the freezing bag, reserving 2 HSC blood samples for each HSC, pasting corresponding bar codes, putting the freezing bag and one blood sample into a precooled freezing storage box (with the corresponding bar codes pasted), transferring the other HSC blood sample into a sample hole, checking that the sample is consistent with the freezing storage box and the freezing bag, transferring the sample and a sample transfer table to the freezing storage chamber, and recording.

Compared with the prior art, the invention has the following beneficial effects: the preparation process is simple, the operation is convenient, and the mechanical damage is reduced; the blood does not leave the sterile environment in the whole process, and the contamination rate is reduced.

Drawings

FIG. 1 is a schematic diagram of the process of extracting placental blood in the method for extracting placental hematopoietic stem cells according to the present invention.

In the figure: 1. villous microvessels; 2. a digestive juice; 3. a catheter with a needle; 4. an umbilical cord; 5. a pump; 6. a triple bag; 7. a pump tube; 8. and (4) connecting the pipes.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

As shown in fig. 1, a method for extracting placental hematopoietic stem cells comprises the following steps:

the method comprises the following steps: the placenta was transported to a laboratory, EP tubes, 5 of which were labeled with maternal blood "m" and 4 of which were labeled with cord blood "Q", were prepared and frozen bags were pasted with bar codes. Taking 1ml and 0.5ml of cord blood whole blood, freezing and storing 1ml in 2 EP tubes, detecting the blood type of 0.5ml, then centrifuging the residual cord blood and maternal blood at 1500rpm/5min, subpackaging, centrifuging the cord blood and subpackaging supernatant, filling the supernatant into 2 EP tubes, wherein each EP tube is 0.5ml, freezing and storing, centrifuging the maternal blood and subpackaging the supernatant, and filling into 4 EP tubes, wherein 3 EP tubes are frozen and stored for rechecking and rechecking, each EP tube is about 0.5ml, 1 EP tube is sent for detection after being subpackaged according to the detection dosage of a single time by a third party, and is used for five infectious diseases and ALT detection; the mother blood cells were transferred to 1 EP tube, frozen and stored together with the mother plasma and cord blood, and the sample storage site was registered.

Step two: washing placenta with NA until the color of the washing solution is light red, and washing placenta at least 15 times.

Step three: the umbilical cord 4 in the placenta is connected with one end of a pump 5 through a pump pipe 7, the other end of the pump 5 is connected with one of the triple bags 6 through the pump pipe 7, the three bags are connected with each other through a connecting pipe 8, the pump 5 is started, the fetal blood distributed in the villus capillaries 1 of the placenta is pumped out and is moved to the triple bag 6.

Step four: connecting the villus microvessels 1 in the placenta with one end of a catheter 3 with a needle, placing the other end of the catheter 3 with the needle in digestive juice 2, starting a pump, and completely cleaning the residual blood in the placenta.

Step five: after cleaning, the pump is closed, and the blood collecting channel of the triple bag 6 is thermally sealed.

Step six: adopting a pressing pulp method to extract the hematopoietic stem cells.

Step seven: the blood bag was mixed on a shaker for 5min and a small portion was shunted into a small transfer bag.

Step eight: placing the uniformly mixed blood bag on a double-face super clean bench, sterilizing the blood bag by using an iodophor cotton ball and an ethanol disinfectant cotton ball, extracting 2.5ml of HSC sample blood from the small transfer bag, subpackaging the blood sample into 3 bar-code-pasted EP tubes, wherein 1.5ml, 0.5ml and 0.5ml are respectively used for rechecking the HSC for standby, and storing the blood sample at-20 ℃; 0.5ml is subpackaged again and is respectively used for cell counting, activity determination and blood type determination; 0.5ml was used for CFU-GM culture, e.g., for typing, and 1.5ml was up-sampled for HLA typing assay.

Step nine: adding HES according to blood volume, sterilizing with iodophor and ethanol disinfectant cotton ball in double-side superclean bench, adding HES into blood bag, and mixing in shaking table for 5 min.

Step ten: the cell number was analyzed by a hemograph.

Step eleven: cell activity assay

Step twelve: the blood bag is put into a large-scale floor type refrigerated centrifuge after being balanced, the temperature is 4-10 ℃, the rotating speed is 480rpm, and the centrifugation is carried out for 6 min.

Step thirteen: and (6) grouting.

Fourteen steps: and (3) putting the blood bag back to the original position in the large floor type refrigerated centrifuge, centrifuging for 5min at the temperature of 4-10 ℃ and the rotating speed of 1500 rpm.

Step fifteen: and (6) grouting.

Sixthly, the steps are as follows: repeating the steps 15-16.

Seventeen steps: after high-speed centrifugation, separating partial plasma in a transfer bag into a sample collection bag by using a plasma separation clamp, controlling 20-40 ml of blood in the transfer bag and keeping the plasma in a blood bag conduit, after heat sealing and shearing off one end of the transfer bag, extracting the plasma in the conduit by using an injector in a clean A-level area, respectively injecting the plasma into 3 EP tubes, storing the plasma together with HSC sample whole blood, maternal blood plasma and maternal blood cells in a refrigerator at 20 ℃ below zero for rechecking, and closing the clamp on the blood bag conduit to heat seal and shear off partial conduit.

Eighteen steps: and taking the heat-sealed sample collecting bag for inoculating the bacteria detection bottle for bacterial culture.

Nineteen steps: mixing the transferring bags containing leucocyte, disinfecting the outer mouth of the blood bag with iodophor and ethanol disinfectant in the A-grade workbench, taking out the concentrated leucocyte by using a 50ml syringe, transferring the concentrated leucocyte into a freezing bag, and simultaneously keeping 0.5ml of blood sample for cell classification counting and CD34+ CD45dim flow cytometry after preparation.

Twenty steps: preparing a cryoprotectant, extracting a proper amount of DMSO and dextran according to the volume of the HSC in the freezing bag and a conversion table to ensure that the final concentration of the DMSO protected cells is 9.8%, uniformly mixing, placing in a refrigerator with the temperature of-20 ℃ for cooling and balancing for 5-15min to below 4 ℃, and simultaneously placing the HSC freezing bag in the refrigerator with the temperature of 4 ℃ for balancing for 15 min.

Twenty one: connecting the tube mouth of the disinfection freezing bag with a corresponding injector according to the bar code number, flatly placing the freezing bag on a horizontal shaking table for fixing, fixing the injector on a 10-channel automatic injection pump, slowly and uniformly injecting the cell freezing protective agent into the freezing bag, and discharging the residual cell freezing protective agent in the injector and the catheter into the HSC, so that the HSC and the cell freezing protective agent are fully mixed, and the cell freezing injury phenomenon is avoided.

Step twenty-two: and (3) heat-sealing off a catheter of the freezing bag, reserving 2 HSC blood samples for each HSC, pasting corresponding bar codes, putting the freezing bag and one blood sample into a precooled freezing storage box, pasting the corresponding bar codes, transferring the other HSC blood sample into a sample hole, checking that the sample is consistent with the freezing storage box and the freezing bag, transferring the sample and a sample transfer table to the freezing storage chamber, and recording.

Claims (1)

1. A method for extracting placenta hematopoietic stem cells comprises the following steps:

the method comprises the following steps: the placenta was transported to a laboratory, EP tubes, 5 of which were labeled with maternal blood "m" and 4 of which were labeled with cord blood "Q", were prepared and frozen bags were pasted with bar codes. Taking 1ml and 0.5ml of cord blood whole blood into 2 EP tubes (1ml is frozen and stored, 0.5ml is used for blood type detection), then centrifuging the residual cord blood and maternal blood at 1500rpm/5min and subpackaging, centrifuging the cord blood and subpackaging supernatant, filling the supernatant into 2 EP tubes, wherein 0.5ml of each EP tube is frozen and stored, centrifuging the maternal blood and subpackaging the supernatant into 4 EP tubes, wherein 3 EP tubes are frozen and stored for rechecking and rechecking, about 0.5ml of each EP tube is used, and 1 EP tube is sent for detection after being subpackaged according to the detection dosage of a single time by a third party and is used for five infectious diseases and ALT detection; the mother blood cells were transferred to 1 EP tube, frozen and stored together with the mother plasma and cord blood, and the sample storage site was registered.

Step two: washing placenta with NA until the color of the washing solution is light red, and washing placenta at least 15 times.

Step three: an umbilical cord (4) in the placenta is connected with one end of a pump (5) through a pump pipe (7), the other end of the pump (5) is connected with one bag in a triple bag (6) through the pump pipe (7), the three bags are connected with each other through connecting pipes (8), the pump (5) is started, fetal blood distributed in villus capillaries (1) of the placenta is extracted, and the fetal blood is moved to the triple bag (6).

Step four: connecting the villus microvessels (1) in the placenta with one end of a catheter (3) with a needle, placing the other end of the catheter (3) with the needle in digestive juice (2), starting a pump, and completely cleaning the residual blood in the placenta.

Step five: after cleaning, the pump is closed, and the blood collecting vessel of the triple bag (6) is thermally sealed.

Step six: adopting a pressing pulp method to extract the hematopoietic stem cells.

Step seven: the blood bag was mixed on a shaker for 5min and a small portion was shunted into a small transfer bag.

Step eight: placing the uniformly mixed blood bag on a double-face super clean bench, sterilizing the blood bag by using an iodophor cotton ball and an ethanol disinfectant cotton ball, extracting 2.5ml of HSC sample blood from the small transfer bag, subpackaging the blood sample into 3 bar-code-pasted EP tubes, wherein the volume of the HSC sample blood sample is 1.5ml, 0.5ml and 0.5ml respectively (1.5 ml is used for ex-warehouse HSC rechecking and is reserved at the temperature of minus 20 ℃, 0.5ml is subpackaged again and is respectively used for cell counting, activity determination and blood type determination, and 0.5ml is used for CFU-GM culture), and if typing is carried out, adding 1.5ml of sample for HLA typing detection.

Step nine: adding HES according to blood volume, sterilizing with iodophor and ethanol disinfectant cotton ball in double-side superclean bench, adding HES into blood bag, and mixing in shaking table for 5 min.

Step ten: the cell number was analyzed by a hemograph.

Step eleven: cell activity assay

Step twelve: the blood bag is put into a large-scale floor type refrigerated centrifuge after being balanced, the temperature is 4-10 ℃, the rotating speed is 480rpm, and the centrifugation is carried out for 6 min.

Step thirteen: and (6) grouting.

Fourteen steps: and (3) putting the blood bag back to the original position in the large floor type refrigerated centrifuge, centrifuging for 5min at the temperature of 4-10 ℃ and the rotating speed of 1500 rpm.

Step fifteen: and (6) grouting.

Sixthly, the steps are as follows: repeating the steps 15-16.

Seventeen steps: after high-speed centrifugation, separating partial plasma in the transfer bag into a sample collection bag (controlling 20-40 ml of blood in the transfer bag) by using a plasma separation clamp, keeping the plasma in a blood bag conduit, after heat-sealing and shearing off one end of the transfer bag, extracting the plasma in the conduit by using an injector in a clean A-level area, respectively injecting the plasma into 3 EP tubes, storing the plasma together with HSC sample whole blood, maternal blood plasma and maternal blood cells in a refrigerator (8 tubes in total) at the temperature of-20 ℃ for rechecking, and closing the clamp on the blood bag conduit to heat-seal and shear off the partial conduit.

Eighteen steps: and taking the heat-sealed sample collecting bag for inoculating the bacteria detection bottle for bacterial culture.

Nineteen steps: mixing the transferring bags containing leucocyte, sterilizing (iodophor and ethanol disinfectant) outer port of blood bag in A-grade workbench, taking out concentrated leucocyte with 50ml syringe, transferring into freezing bag, and collecting blood sample 0.5ml for cell classification and counting and CD34+ CD45dim flow cytometry after preparation.

Twenty steps: preparing a cryoprotectant, extracting a proper amount of DMSO and dextran according to the volume of the HSC in the freezing bag and a conversion table to ensure that the final concentration of the DMSO protected cells is 9.8%, uniformly mixing, placing in a refrigerator with the temperature of-20 ℃ for cooling and balancing for 5-15min to below 4 ℃, and simultaneously placing the HSC freezing bag in the refrigerator with the temperature of 4 ℃ for balancing for 15 min.

Twenty one: connecting the tube mouth of the disinfection freezing bag with a corresponding injector according to the bar code number, flatly placing the freezing bag on a horizontal shaking table for fixing, fixing the injector on a 10-channel automatic injection pump, slowly and uniformly injecting the cell freezing protective agent into the freezing bag, and discharging the residual cell freezing protective agent in the injector and the catheter into the HSC, so that the HSC and the cell freezing protective agent are fully mixed, and the cell freezing injury phenomenon is avoided.

Step twenty-two: and (3) heat-sealing off a catheter of the freezing bag, reserving 2 HSC blood samples for each HSC, pasting corresponding bar codes, putting the freezing bag and one blood sample into a precooled freezing storage box (with the corresponding bar codes pasted), transferring the other HSC blood sample into a sample hole, checking that the sample is consistent with the freezing storage box and the freezing bag, transferring the sample and a sample transfer table to the freezing storage chamber, and recording.

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