Stem Cell Preservation and Resuscitation
Stem Cell Preservation and Resuscitation
Hematopoietic stem cell transplantation is an effective treatment for malignant hematological disorders, oncological radiotherapy and severe radiation sickness Q However, severe graft-versus-host disease (GVHD) often occurs after allogeneic stem cell transplantation due to the presence of histocompatibility antigens leading to graft failure. Finding hematopoietic stem cells that exactly match the patient's histocompatibility antigens is very difficult and takes a long time; for this reason, carrying out stem cell preservation is an important measure to make such cells available for patient transplantation on an elective basis. Author: Xuedao Pei, this experiment is from "Stem cell experiment guide".
Operation method
Preservation and Recovery of Stem Cells Move I. Cryopreservation methods for hematopoietic stem cells Cryopreservation methods for fetal liver hematopoietic stem cells 1 ) Preparation of fetal liver cell suspension Human fetal liver was obtained from a normal 4-6 month old fetus that had been induced by hydrosalpinx or cesarean section. The fetal liver was removed from the abdominal cavity under aseptic conditions, and blood was taken from the heart to check the fetal blood group. After removing the connective tissue and gallbladder adhering to the surface of the liver, the liver was placed in a Petri dish and cut into small pieces of tissue, which were then put into a small beaker with a double-layer stainless steel (the inner pore size of 1,000 mesh, and the outer pore size of 1,800 mesh/in2 ( lin2=6. 451600xl0-4 m 2 )). With a small amount of culture medium or saline (after cell culture, the pyrogen test is negative), the tissue block was rinsed 2 ~ 3 times, discard the washing solution, and then poured into the 20 ml P R M I 1640 culture medium, and the syringe needle core in the stencil to grind the tissue block, and so on 3 times, can be removed from the majority of hematopoietic stem cells in the fetal liver. The combined collected cell suspensions were passed through a 4-gauge needle syringe to make a single-cell suspension. 2 ) Addition of cryoprotective solution Prepare a sterile protective solution (DMS0 ) to a concentration of 20 % with 1640 culture medium. Take 20 % D M S O in the same volume as the fetal liver suspension and slowly add it to the cell suspension, let it stand for 10 min at room temperature, and then put the sample into the cryostat. 3) Cooling method Program cooling method: Most of the two-step or three-step cooling method, that is, at the beginning of the room temperature to reduce the temperature, down to -30 ℃, a constant temperature of 2m i n after the change to 5-7 ℃ / m i n down to -80 ℃ or -90 ℃, and then transferred to liquid nitrogen (- 196 ℃) for long-term preservation. Some people also advocate that the cooling rate should be strictly controlled at 1-3°C/min at the beginning, but after the heat release of phase change (-11-15°C), the cooling rate can be accelerated, and the remaining phases have little effect on the survival of cells (Liu Jingang 1993). 1 ) Bone Marrow Collection Bone marrow should be performed aseptically in the operating room. Under local or general anesthesia, multiple punctures are made anteriorly and posteriorly with a bone marrow aspiration needle to extract the bone marrow. The syringe used to extract the bone marrow should be heparinized, and the amount of heparin per milliliter of bone marrow should be controlled to 20 - 40 units to prevent the formation of agglutination. When patients with malignant blood diseases are mobilized with CMP chemotherapy combined with rhG-CSF (l0ng/kg per day), and the white blood cells rise to more than 5xl09 cells/L, PBSC is collected by CS-3000P LUS hematocrit machine, and the total amount of blood circulation processed is 7,000-16,000ml (set according to the patient's condition), and the total output is 55-60 ml. The total amount of blood circulation processed is 7000-16OOOml (set according to the patient's condition), and the total output is 55-60 ml. At present, there are two main methods for in vitro preservation of peripheral blood hematopoietic stem cells: programmed liquid nitrogen cryopreservation at -196°C and direct preservation at -80°C. The former method is widely used at home and abroad because of its long-term preservation and low cell loss. The former method is widely used at home and abroad as a classic method for long-term preservation with low cell loss (Almid 1997, Broxmeyer 1997), but the disadvantages are complicated operation and expensive equipment. - The method of direct freezing at 80℃ was developed from the 1980s of the 20th century, and its advantages are simple operation, low cost and practical, but there are fewer reports of long-term hematopoietic stem cell preservation studies using this method, therefore, it is currently considered that direct freezing at -80℃ is suitable for the more Program-controlled cooling means that the cooling process is controlled by a program, and after the program is finished, the PBSC is put into liquid nitrogen for preservation. At this time, all the enzyme activities are inhibited, and the rays in the environment will not cause ionization damage to the cells, so it can be preserved for a longer period of time. This technique uses only dimethyl sulfoxide (DMSO) as cryoprotective fluid and is the classic method of hematopoietic stem cell preservation. (1) Preparation of cryoprotective liquid: 50% RPMI 1640 nutrient solution, 20% DMSO, 30% human AB serum or autologous plasma. (2) Adjust the cell concentration of the collected PBSC with RPMI 1640 nutrient solution in the range of 2 x l0 7 - l x l0 8, and mix with equal amount of RPMI 1640 nutrient solution. (3) The freezer bag (tube) into the pre-cooled to 4 ℃ program-controlled cooling instrument, programmed cooling. -l ℃ / min, down to -40 ℃, equilibrate for 3 min, -5 ℃ / min, down to -80 ℃, remove and put into liquid nitrogen (-196 ℃) storage. The storage time is 5 years. (4) P B S C resuscitation and re-infusion: frozen storage from the liquid nitrogen removed immediately into the 42 ℃ water bath box, completely melted immediately after rapid re-infusion to the patient. (5) Determination of cell viability: The samples to be tested were examined for cell viability before cryopreservation and after resuscitation at Oh, 3 h, and 6 h, respectively, by using the Taipan blue refractory staining method (Xiu-Rong Lv 2 0 0 4 ). This is the freezing method without speed control condition, which is simple, reliable and easy to repeat. However, due to the fact that the cooling rate of this cooling method is not constant during the cooling process, the choice of cryoprotectant and its effect on cell recovery has been highly emphasized (Peng, W. J. 2003). 1 ) Cryoprotective fluid Stiff reported a method of freezing bone marrow cells at -80 °C by unprogrammed cooling using a mixture of 6 % hydroxyethyl starch (HES), 5 % DMSO and 4 % human serum albumin (ALB) as cryoprotective solution. Halle et al. (2004) used 1 % H A S + 3 . 5 % H E S + 2 . Halle et al. (2004) used 1 % HAS + 3.5 % HES + 2.5 % DMSO. Zhang Junping et al. used different ratios of DMSO, HES, ALB and HAS to form a cryoprotective solution, and found that 5 % DMSO + 6 % HES + HAS was the most effective. Fan Hua Hua et al. developed CP80 cryoprotective liquid containing 7.5% dextran, 6.25% DMSO and 6.25% ALB, and the concentration of DMSO was 5 % after mixing with the sample. A single cryoprotectant has also been used. Galmes et al. (1995) first proposed the use of 10% DMSO as a protective solution and storage of cells at -80°C after freezing. Hemandez-Navarro et al. (1995) used a mixture of 10% D M S O and H A S as cryoprotective solution, and the frozen bags filled with P B S C were put into 95% methanol solution and stored at -80°C directly, and the hematopoietic recovery of the cells after transfusion did not differ significantly from that of other methods. Ye Yunbin et al. (2000) compared the effects of using 10% DMSO single protective solution and 5 % DMSO + 6 % HES + 4 % ALB mixture, and found that there was no significant difference between the two within 180 days of cryopreservation, with cell viability above 85 %, and the proportion of CD 34+ positive cells and the number of monoclonal colonies maintained at the therapeutic level. 2 ) Cell concentration A cell concentration of 0. 5xl08 - 1. 0xl08 cells/m l is commonly used for clinical freezing of PBSC. McElwain, Mangalik, and Bumett et al. have successfully stored PBSCs at 4°C for 8 h, 72 h, and 48 h, respectively, and then re-infused them. Non-cryogenic 4 ¾ preservation of P B S C simplifies the transplantation process, eliminates the need to add cryoprotective fluid, avoids the damage to the cells during the freezing process, and only requires bone marrow maintenance fluid, which reduces the toxic side effects of re-infusion; reduces the chances of in vitro contamination of P B S C; and is conducive to hematopoietic reconstruction. Therefore, it is a simple, feasible, safe, reliable, cost-effective method of in vitro preservation. The disadvantage is that the preservation time is short, not suitable for more than 72 h, when the pretreatment program for 3 ~ 4 days. 1) Umbilical cord blood collection Umbilical cord blood was collected from newborns delivered by normal delivery or cesarean section by closed collection method. Pregnant women were aged less than 35 years, had a full-term pregnancy, had no complications, had a prenatal blood test for Hb >90 g/L, and were negative for HbsAg, HCV, anti-HIV1/2 antibodies and syphilis. The mother and husband had no family history of hereditary diseases. The blood was collected from the severed end of the umbilical cord away from the newborn before delivery of the placenta in a closed blood bag anticoagulated with sodium citratephosp ate dextrose adenine (CPDA). The blood was stored at room temperature after collection and transported to the cord blood bank within 18 hours. 2 ) Umbilical cord blood isolation Meter with 6 % (mass fraction) light ethyl starch (h e s , average relative molecular mass 480 000, H e s p a n , D u p o n , U.S.A.) sedimentation and centrifugation method to remove most of the red blood cells and most of the plasma. Specific method: each part of cord blood according to the volume ratio of 1 : 4 ratio added to the cord blood, fully mixed, vertical inverted suspension, so that the natural settlement of red blood cells 20 ~ 30 m i n , until the red blood cell interface no longer decline, remove the red blood cells. The nucleated cell-rich blood was then centrifuged at 100°C 4 00g for l O m i n , and the nucleated cells were settled. A plasma accumulator was used to remove some of the plasma. The volume of plasma was 30-40 ml. The above operations were performed in a collection bag (Kaiyan Liu 2003). 3) Umbilical cord blood direct preservation Programmed cooling deep cryopreservation method Protective solution: 20% Dimethyl Sulfoxide (DMSO, Sigma), 80% TCE. In an ice-water bath, the concentrated nucleated cells were added to an equal volume of cryoprotective solution (final concentration of 10% DMSO), mixed well, and the samples were put into a DW microcomputer cooling instrument, cooled down to -40 ℃ at a rate of 0.001/million, and then cooled down to -80 ℃ at a rate of 5T/million, and then taken out and stored in liquid nitrogen, and the final concentration of nucleated cells was (2.34±2.06) xl09 cells/L (Ma Q. 2003). The final concentration of nucleated cells was (2.34±2.06)xl09 cells/L (Qing Ma 2003). Non-programmed cooling deep cryopreservation method The following is a description of several preservation methods for different protective fluid formulations: (1) Protective solution formula :20 % D M S O , 12 % dextran (dextmn, Sigma), physiological sodium chloride solution. In an ice-water bath, the concentrated nucleated cells were added to an equal volume of cryoprotective solution (DMSO final concentration of 10%), mixed well, and stored in a refrigerator at -80℃ for 4 h, then stored in liquid nitrogen, and the final concentration of nucleated cells was (2.34±2.06) xl09 cells/L (Ma Q. 2003). (2) 16-milk culture solution of equal volume with cord blood, 10 ml of ACD culture solution, 20% DMSO, mixed well, slowly added into cord blood (the final concentration of DMSO was 10%), stood still for 5 min, put into liquid nitrogen gas phase, and put into liquid nitrogen after 30 m i n . (3) Protective liquid formula: 10% DMSO, 12% dextrose liver, 5% albumin, physiological sodium chloride solution. In an ice-water bath, add the concentrated nucleated cells to an equal volume of cryoprotective solution (DMSO final concentration of 5%), mix well, and store in a refrigerator at -80 ℃, with a final concentration of nucleated cells of (2.34±2.06)xl09 cells/L (Ma Qing 2 0 0 3). There are various forms of rewarming due to different methods of preservation. 1 ) Without removing the protective fluid Remove the sample from liquid nitrogen and rewarm it quickly in a water bath at 37-42°C for no more than 5 min. Filter the cord blood and place it in a sterilized infusion bottle, seal it, and wait for the patient to use it. 2 ) Removal of protective agent The samples were rewarmed in a water bath at 41 ℃, and each sample was frozen and thawed within 2 m i n. The samples were equilibrated with 5 % albumin and 10% L M D for 5 min. Add equal volumes of 5% albumin and 10% LMD, mix and equilibrate for 5 min, then centrifuge to remove the DMSO, and adjust the cell suspension with LMD (Guangwu Li 1998). For more product details, please visit Aladdin Scientific website.
Cryopreservation of peripheral blood hematopoietic stem cells
Therefore, it is currently considered that -80℃ direct freezing is suitable for shorter time of hematopoietic stem cell freezing. Cryoprotective liquid is one of the keys to the effect of freezing.
The collected P B S C was mixed with an equal amount of pre-cooled cryoprotective solution and injected into the cryobag separately, and the test samples were reserved for injection into the cryotubes.
Umbilical cord blood retrieved from hospitals in the authors' laboratory was directly added to an equal volume of protective fluid without any treatment, and liquid nitrogen preservation achieved better results.