CN112899221A - Method for processing disease-type hiPSC-CM cells for real-time cell analysis technology - Google Patents
Method for processing disease-type hiPSC-CM cells for real-time cell analysis technology Download PDFInfo
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Abstract
The invention discloses a method for processing disease-type hipSC-CM cells for a real-time cell analysis technology. The treatment method comprises the steps of thawing, slowly dripping the myocardial cell resuscitation solution and the like, and the steps are detailed in the invention. The treatment method of the invention greatly improves the survival rate of disease-type hiPSC-CM cells and shortens the culture time.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for processing disease type hipSC-CM cells for a real-time cell analysis technology.
Background
The Human induced pluripotent stem cell-derived cardiac cytocyte (hiPSC-CM) is a Human-derived cardiomyocyte which is derived from autologous cells, is formed by utilizing a reprogramming technology and directional differentiation, has the same genotype as a patient donor, and is widely applied to the evaluation research of drug in-vitro cardiotoxicity. Compared with other myocardial cell models, the disease-type hiPSC-CM is fragile and easily influenced by external condition factors, and when the recovery and treatment method is used for real-time cell analysis and detection, the recovery and treatment method is very important for the stability of the cell pulsation frequency and amplitude. The conventional hiPSC-CM resuscitation method in the field adopts a culture medium to be added dropwise for inoculation and culture after thawing cryopreserved cells, so that the conventional resuscitation treatment method cannot meet the requirement of practical application because the conventional resuscitation treatment method usually causes more myocardial cell death number, slower cell index increase, later cell pulsation time point and unstable pulsation frequency and amplitude, and researchers are dedicated to developing new culture methods due to the call of the novel disease-type hiPSC-CM resuscitation and treatment methods with higher survival rate and more stable cell state in the field.
In addition, due to the differences in physiological properties between disease-type hiPSC-CM cells and healthy hiPSC-CM cells, different seeding densities have a large impact on their pulsatile stability. Cells with too low seeding density cannot beat, and cells with too high seeding density affect the stability and uniformity of cell beating, so that the determination of proper seeding density is an important step for RTCA experiments.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for treating disease-type hiPSC-CM cells for real-time cell analysis technology in order to overcome the defects that only a recovery and culture method of healthy hiPSC-CM cells is provided in the prior art and the pulsation performance of the cells obtained by the existing treatment method is poor.
In order to solve the above problems, one of the technical solutions adopted by the present invention is: a method for processing disease-type hiPSC-CM cells for real-time cell analysis technology, comprising the following resuscitation steps:
(1) thawing diseased hiPSC-CM cells to be revived;
(2) adding the resuscitation solution 1 into the thawed disease-type hiPSC-CM cells at a speed of 4 seconds per drop; adding the resuscitation solution 2 into the thawed disease-type hiPSC-CM cells dropwise from the speed of 2 drops per second to 2 drops per second; the volume ratio of the resuscitation solution 1 to the thawed diseased hiPSC-CM cells is 1.5: 1-3: 1, and the volume ratio of the resuscitation solution 2 to the thawed diseased hiPSC-CM cells is 8: 1-10: 1; the resuscitation liquid 1 and the resuscitation liquid 2 are cardiac muscle cell resuscitation liquids.
Preferably, the volume ratio of the resuscitation fluid 1 in step (2) to the thawed diseased hiPSC-CM cells is 2: 1.
Preferably, the cardiomyocyte resuscitation solution in step (2) is purchased from Nanjing El & lt & gtregenerative medicine science and technology Co., Ltd, Cat No: HELP 4002.
Preferably, the thawing in step (1) comprises: putting the disease type hiPSC-CM to be resuscitated into a water bath kettle at 37 ℃, taking out the disease type hiPSC-CM after shaking for 1-2 minutes, wiping the surface of the cryopreservation tube with 75% alcohol, and transferring the tube to a biological safety cabinet.
Preferably, the method for processing disease-type hiPSC-CM cells for real-time cell analysis further comprises the following steps:
(3) centrifuging at room temperature for 4-6 min at 200-400 g, adding a cardiac muscle cell recovery liquid, and gently blowing and beating cells to prepare a cell suspension; adding cell suspension into the coated RTCA E-Plate Cardio 96 pore Plate to make the number of cells per pore Plate be (6-7) × 104Mixing, placing into RTCA Cardio workstation, and changing the solution every 48 hr.
Preferably, the RTCA E-Plate Cardio 96-well Plate is coated with a plating solution, preferably 0.1% gelatin or 10mg/mL fibronectin; more preferably from Nanjing El & lt & gtProv, regenerative medicine science and technology Limited, Cat No.: HELP 4004.
Preferably, the speed of the centrifugation in the step (3) is 300g, and the time of the centrifugation is 5 min.
Preferably, the number of cells per well in step (3) is 6X 104Or 7 x 104And (4) respectively.
Preferably, the method for processing disease-type hiPSC-CM cells for real-time cell analysis technology further comprises system self-inspection.
Preferably, the system self-test comprises the following steps: adding 50 mu L of myocardial cell plating solution into each hole of an RTCA E-Plate Cardio 96 pore Plate, standing at 37 ℃ for 30min, taking out, adding 50 mu L of myocardial cell resuscitation solution into each hole, and placing at an RTCA Cardio workstation for detecting a baseline.
Preferably, the method for processing disease-type hiPSC-CM cells for real-time cell analysis technology comprises the following steps:
(1) adding 50 mu L of myocardial cell plating solution into each hole of an RTCA E-Plate Cardio 96 pore Plate, standing at 37 ℃ for 30min, taking out, adding 50 mu L of myocardial cell resuscitation solution into each hole, and placing at an RTCA Cardio workstation for detecting a baseline;
(2) taking out disease type hiPSC-CM to be resuscitated from a liquid nitrogen tank, quickly placing the lower 1/2 of the cryopreserved pipe in a water bath kettle at 37 ℃, shaking gently for 1-2 minutes until the pipe is almost completely thawed, taking out, wiping the surface of the cryopreserved pipe with 75% alcohol, and transferring the cryopreserved pipe to a biological safety cabinet; transferring the liquid in the cryopreservation tube to a 50mL centrifuge tube by using a 1mL pipette, gently flushing the cryopreservation tube by using 2mL resuscitation liquid, slowly adding flushing liquid into the 50mL centrifuge tube at a speed of 4 seconds per drop, and gently shaking the centrifuge tube while dropping to gradually dilute the flushing liquid; adding 3-5 mL of resuscitation solution into a centrifuge tube at a speed of 2 seconds per drop, and adding 6-10 mL of resuscitation solution into the centrifuge tube dropwise from a high speed (2 seconds per drop) to a low speed (1 second 2 drops) for counting; where "near complete melting" is described as melting to an ice-water mixture, where only a very small amount of ice remains.
(3) Centrifuging for 5min at room temperature of 300g, adding appropriate amount of myocardial cell resuscitating solution, gently blowing and beating cells to prepare cell suspension, adding 100-hole cell suspension into each hole of the coated RTCA E-Plate Cardio 96 pore Plate to make the number of cells in each hole be (6-7) x 104And the mixture is put into an RTCA Cardio workstation after being shaken and mixed uniformly, and is changed into the myocardial cell culture solution after 48 hours, and the solution is changed every 48 hours.
Preferably, the cardiomyocyte culture medium is purchased from Nanjing El & lt & gt regenerative medicine science and technology, Cathaka: HELP 4001.
The numbers "1" and "2" following a term in the present invention have no practical meaning, but merely distinguish the same term. For example, the numbers after "resuscitation fluid 1" and "resuscitation fluid 2".
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention. The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the survival rate of disease-type hiPSC-CM cells treated by the treatment method is higher than 65%; the disease type hiPSC-CM cells obtained by the treatment method can record continuous pulsation signals of the cells at the earliest time within 32 hours, and can obtain the cardiomyocytes which have the qualified cell activity and are suitable for follow-up research within about 110 hours, wherein the pulsation frequency of the cardiomyocytes which have the qualified cell activity is higher than 32 +/-3 (1/min), the pulsation amplitude is (0.04-0.05) +/-0.01, and the cell pulsation irregularity index is below 40%. Therefore, the present invention greatly improves the survival rate of disease-type hiPSC-CM cells and shortens the culture time.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Disease-type cardiomyocytes, cardiomyocyte plating solution, cardiomyocyte resuscitation solution (also called resuscitation solution for short) and cardiomyocyte culture solution are purchased from Nanjing El's regenerative medicine science and technology Limited.
Example 1
(1) Adding 50 μ L of cardiomyocyte plating solution (HELP 4004, Nanjing El medicine science and technology Co., Ltd.) into each well of RTCA E-Plate Cardio 96 well Plate, standing at 37 deg.C for 30min, taking out, sucking away the plating solution, adding 50 μ L of cardiomyocyte resuscitation solution (HELP 4002, Nanjing El medicine science and technology Co., Ltd.) into each well, and placing in RTCA Cardio workstation to detect baseline.
(2) Taking out the disease type hiPSC-CM to be resuscitated from the liquid nitrogen tank, quickly placing the lower 1/2 of the cryopreservation tube in a water bath kettle at 37 ℃, shaking gently for 1-2 minutes until a small piece of ice remains, taking out, wiping the surface of the cryopreservation tube with 75% alcohol, and transferring to a biological safety cabinet. Transfer of the liquid in the vial to a 1mL pipetteA 50mL centrifuge tube, wherein the cryopreserving tube is gently flushed by 2mL resuscitation solution, the flushing solution is slowly added into the 50mL centrifuge tube at a speed of 4 seconds per drop, and the centrifuge tube is gently shaken while dropping is carried out to gradually dilute the flushing solution; adding 3-5 mL of resuscitation solution into the centrifuge tube at a speed of 2 seconds per drop, dropwise adding 6-10 mL of resuscitation solution into the centrifuge tube at a speed of 1 drop in 2 seconds to 1 drop in 1 second, counting, and counting to obtain a dead cell number of 180.9 multiplied by 104The number of whole cells was 567.0X 104Number of viable cells is 386.1X 104The activity rate is 68.1%.
(3) Centrifuging at room temperature for 5min at 300g, adding appropriate amount of resuscitating solution, gently blowing and beating cells to prepare cell suspension, adding 100 μ L of cell suspension into coated RTCA E-Plate Cardio 96 well Plate to make the number of cells in each well 7 × 104The whole is put into an RTCA Cardio workstation after cross shaking and mixing, and is changed into a myocardial cell culture solution (HELP 4001, manufactured by Nanjing El's regenerative medicine science and technology Co., Ltd.) after 48 hours, and the solution is changed every 48 hours. The continuous pulsation signal of the cell is recorded at the earliest 32h, the myocardial cell which reaches the cell viability standard and is suitable for subsequent research can be obtained at about 110h, the cell pulsation frequency is 38 +/-5 (1/min), the pulsation amplitude is 0.04 +/-0.01, and the cell pulsation irregularity index is below 40 percent.
Example 2
(1) Adding 50 μ L of cardiomyocyte plating solution (HELP 4004, Nanjing El medicine science and technology Co., Ltd.) into each well of RTCA E-Plate Cardio 96 well Plate, standing at 37 deg.C for 30min, taking out, sucking away the plating solution, adding 50 μ L of cardiomyocyte resuscitation solution (HELP 4002, Nanjing El medicine science and technology Co., Ltd.) into each well, and placing in RTCA Cardio workstation to detect baseline.
(2) Taking out the disease type hiPSC-CM to be resuscitated from the liquid nitrogen tank, quickly placing the lower 1/2 of the cryopreservation tube in a water bath kettle at 37 ℃, shaking gently for 1-2 minutes until a small piece of ice remains, taking out, wiping the surface of the cryopreservation tube with 75% alcohol, and transferring to a biological safety cabinet. Transferring the liquid in the cryopreservation tube to a 50mL centrifuge tube by using a 1mL pipette, gently flushing the cryopreservation tube by using 2mL resuscitation solution, slowly adding flushing liquid into the 50mL centrifuge tube at a speed of 4 seconds per drop, and gently shaking the centrifuge tube while dropping to enable the centrifuge tube to be in a state of droppingGradually diluting; adding 3-5 mL of resuscitation solution into a centrifuge tube at a speed of 2 seconds per drop, dropwise adding 6-10 mL of resuscitation solution into the centrifuge tube at a speed of from slow (1 drop in 2 seconds) to fast (2 drops in 1 second), counting, wherein the number of dead cells is 187.2 multiplied by 104The number of whole cells was 529.2X 104Number of viable cells was 342.0X 104The activity rate is 64.6%.
(3) Centrifuging at room temperature for 5min at 300g, adding appropriate amount of resuscitating solution, gently blowing and beating cells to prepare cell suspension, adding 100 μ L of cell suspension into coated RTCA E-Plate Cardio 96 well Plate to make the number of cells in each well 6 × 104The whole is put into an RTCA Cardio workstation after cross shaking and mixing, and the culture solution is changed into a myocardial cell culture solution (HELP 4001, SeiyingAirlpu regenerative medicine science and technology Co., Ltd.) after 48 hours, and the solution is changed every 48 hours. The continuous pulsation signal of the cell is recorded at the earliest time of 38h, the myocardial cell which reaches the cell viability standard and is suitable for subsequent study can be obtained at about 134h, the cell pulsation frequency is 32 +/-3 (1/min), the pulsation amplitude is 0.05 +/-0.01, and the cell pulsation irregularity index is basically below 40 percent.
Comparative example 1
(1) Adding 50 μ L of cardiomyocyte plating solution (HELP 4004, Nanjing El medicine science and technology Co., Ltd.) into each well of RTCA E-Plate Cardio 96 well Plate, standing at 37 deg.C for 30min, taking out, sucking away the plating solution, adding 50 μ L of cardiomyocyte resuscitation solution (HELP 4002, Nanjing El medicine science and technology Co., Ltd.) into each well, and placing in RTCA Cardio workstation to detect baseline.
(2) Taking out the disease type hiPSC-CM to be resuscitated from the liquid nitrogen tank, quickly placing the lower 1/2 of the cryopreservation tube in a water bath kettle at 37 ℃, shaking gently for 1-2 minutes until a small piece of ice remains, taking out, wiping the surface of the cryopreservation tube with 75% alcohol, and transferring to a biological safety cabinet. Transferring the liquid in the cryopreserving tube to a 50mL centrifuge tube by using a 1mL pipette, gently flushing the cryopreserving tube by using 1mL resuscitation solution, and slowly dropwise adding the flushing liquid into the 50mL centrifuge tube, wherein the process is completed within about 20 seconds; adding the resuscitating solution of 2-10 mL into the centrifuge tube drop by drop from slow to fast, completing the resuscitating process within 5 minutes, counting, and obtaining the number of dead cells of 292.5 multiplied by 104The number of whole cells was 594.0X 104Number of viable cells is 301.5X 104The activity rate is 50.8%.
(3) Centrifuging at room temperature for 5min at 300g, adding appropriate amount of resuscitating solution, gently blowing and beating cells to prepare cell suspension, adding 100 μ L of cell suspension into coated RTCA E-Plate Cardio 96 well Plate to make the number of cells in each well 5 × 104The whole is put into an RTCA Cardio workstation after cross shaking and mixing, and the culture solution is changed into a myocardial cell culture solution (HELP 4001, SeiyingAirlpu regenerative medicine science and technology Co., Ltd.) after 48 hours, and the solution is changed every 48 hours. The continuous pulsation signal of the cell is recorded at the earliest time of 97h, the myocardial cell which reaches the cell viability standard and is suitable for subsequent research can be obtained at about 197h, the cell pulsation frequency is 44 +/-9 (1/min), the pulsation amplitude is 0.03 +/-0.02, and the cell pulsation irregularity index is basically below 80 percent.
Claims (10)
1. A method for processing disease-type hiPSC-CM cells for real-time cell analysis technology, comprising the following resuscitation steps:
(1) thawing diseased hiPSC-CM cells to be revived;
(2) adding the resuscitation solution 1 into the thawed disease-type hiPSC-CM cells at a speed of 4 seconds per drop; dropwise adding the resuscitation solution 2 into the thawed disease-type hiPSC-CM cells at a speed of 1 drop for 2 seconds to 2 drops for 1 second; the volume ratio of the resuscitation solution 1 to the thawed diseased hiPSC-CM cells is 1.5: 1-3: 1, and the volume ratio of the resuscitation solution 2 to the thawed diseased hiPSC-CM cells is 8: 1-10: 1; the resuscitation liquid 1 and the resuscitation liquid 2 are cardiac muscle cell resuscitation liquids.
2. The treatment method of claim 1, wherein the volume ratio of resuscitation fluid 1 to thawed diseased hiPSC-CM cells in step (2) is 2: 1.
3. The process of claim 1, wherein the cardiomyocyte resuscitation solution used in step (2) is purchased from Nanjing Airlop regenerative medicine science and technology, Inc., Cat #: HELP 4002.
4. The process of claim 1, wherein said thawing of step (1) comprises: putting the disease type hiPSC-CM to be resuscitated into a water bath kettle at 37 ℃, taking out the disease type hiPSC-CM after shaking for 1-2 minutes, wiping the surface of the cryopreservation tube with 75% alcohol, and transferring the tube to a biological safety cabinet.
5. The process of any one of claims 1 to 4, further comprising the steps of:
(3) centrifuging at room temperature for 4-6 min at 200-400 g, adding a cardiac muscle cell recovery liquid, and gently blowing and beating cells to prepare a cell suspension; adding cell suspension into the coated RTCA E-Plate Cardio 96 pore Plate to make the number of cells per pore Plate be (6-7) × 104Uniformly mixing, putting into an RTCA Cardio workstation, and changing the solution every 48 hours; preferably, the RTCA E-Plate Cardio 96-well plates are coated with a plating solution, preferably 0.1% gelatin or 10mg/mL fibronectin, more preferably from tokyo elpfu regenerative medicine science co. HELP 4004.
6. The process according to claim 5, wherein the speed of the centrifugation in step (3) is 300g and the time of the centrifugation is 5 min.
7. The process of claim 5, wherein the number of cells per well in step (3) is 6X 104Or 7 x 104And (4) respectively.
8. The process of any one of claims 1 to 7, further comprising a system self-test.
9. The process of claim 8, wherein the system self-test comprises the steps of: adding 50 mu L of myocardial cell plating solution into each hole of an RTCA E-Plate Cardio 96 pore Plate, standing at 37 ℃ for 30min, taking out, adding 50 mu L of myocardial cell resuscitation solution into each hole, and placing at an RTCA Cardio workstation for detecting a baseline.
10. The process according to any one of claims 1 to 9, characterized in that it comprises the following steps:
(1) adding 50 mu L of myocardial cell plating solution into each hole of an RTCA E-Plate Cardio 96 pore Plate, standing at 37 ℃ for 30min, taking out, adding 50 mu L of resuscitation solution into each hole, and placing at an RTCA Cardio workstation for detecting a baseline;
(2) taking out the disease type hiPSC-CM to be resuscitated from the liquid nitrogen tank, quickly placing the lower 1/2 of the cryopreserved pipe in a water bath kettle at 37 ℃, shaking gently for 1-2 minutes until the cryopreserved pipe is almost completely melted, taking out the cryopreserved pipe when the cryopreserved pipe is nearly completely melted, wiping the surface of the cryopreserved pipe with 75% alcohol, and transferring the cryopreserved pipe to a biological safety cabinet; transferring the liquid in the cryopreservation tube to a 50mL centrifuge tube by using a 1mL pipette, gently flushing the cryopreservation tube by using 2mL resuscitation liquid, slowly adding flushing liquid into the 50mL centrifuge tube at a speed of 4 seconds per drop, and gently shaking the centrifuge tube while dropping to gradually dilute the flushing liquid; adding 3-5 mL of resuscitation solution into a centrifuge tube at a speed of 2 seconds and 1 drop, dropwise adding 6-10 mL of resuscitation solution into the centrifuge tube from 2 seconds and 1 drop to 1 second and 2 drops, and counting;
(3) centrifuging for 5min at room temperature of 300g, adding a proper amount of resuscitation solution, gently blowing and beating cells to prepare cell suspension, adding 100-hole cell suspension into each coated RTCA E-Plate Cardio 96-well Plate to enable the number of cells in each hole to be (6-7) x 104The mixture is put into an RTCA Cardio workstation after being shaken and mixed in a cross way, and is changed into a myocardial cell culture solution after 48 hours, and the solution is changed every 48 hours; preferably, the cardiomyocyte culture medium is purchased from Nanjing El & lt & gt regenerative medicine science and technology, Cathaka: HELP 4001;
the resuscitation solution is a cardiac muscle cell resuscitation solution.
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