CN114807018B - Passage method of pluripotent stem cells - Google Patents
Passage method of pluripotent stem cells Download PDFInfo
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Abstract
The invention discloses a passage method of pluripotent stem cells, which comprises a passage tool and prepared dissociation liquid; the passage tool comprises a precise spring, a rolling shaft and tweezers, the precise spring is sleeved on the rolling shaft, a clamping groove is formed in the surface of the rolling shaft and can be fixed at the tip of the tweezers through a compression spring to adjust the distance between spring wires, the assembly can be used for passage of the pluripotent stem cells after sterilization, the tweezers are held to push and roll the precise spring during passage, the pluripotent stem cell colonies are cut by the precise spring wires and cut into small pieces with uniform sizes, then dissociation liquid is added to enable the cut pluripotent stem cell pieces to be easily eluted and collected by pipetting, and further the passage expansion of the pluripotent stem cells is completed. The method is simple and reliable, is universal and easy to operate, has low cost, integrates various advantages of the existing method, does not need digestion by enzymes and calcium ion chelating agents, does not damage extracellular matrixes of the pluripotent stem cells, and can efficiently carry out passage of the pluripotent stem cells.
Description
Technical Field
The invention belongs to the technical field of stem cell passage, and particularly relates to a passage method of pluripotent stem cells.
Background
The pluripotent stem cells have the capability of self-renewal and multidirectional differentiation, are the basis of drug screening, tissue and organ regeneration and medical treatment, and have great application value; pluripotent stem cells are usually grown on mouse feeder cells or special colloid coated dishes by adherence and spread to form typical circular, polygonal colonies; when the colony is close to fusion, the pluripotent stem cells need to be subjected to passage expansion; the common amplification mode is that enzyme digestion, such as type IV collagenase, dispase and the like, is adopted, the treatment time is 5-10 minutes, after the edges of the pluripotent stem cell colonies are slightly rolled up, a certain amount of culture medium is added for cleaning, a pipetting gun or a pipette is used for blowing and sucking the culture medium, and the pluripotent stem cell colonies are blown and scattered into clusters of 10-30 stem cells, and are transferred into a new culture dish for amplification and growth; the size of the agglomerate cannot be effectively controlled by blowing, the excessive blowing can easily cause spontaneous differentiation of the next generation of pluripotent stem cells, the excessive blowing can easily cause apoptosis after passage of the pluripotent stem cells, and the agglomerate with different sizes can not synchronize the growth of the pluripotent stem cells and cannot meet the differentiation and medicine requirements homogeneously; the capability of forming a cell mass that effectively controls the size of pluripotent stem cells at passage is one of the key techniques for research and application of pluripotent stem cells.
Currently, invitrogen corporation in the United states provides a tool StemPro EZPassage specially used for passaging pluripotent stem cells, which can vertically and crosswise cut pluripotent stem cell colonies into small pieces with uniform size by using specially prepared rollers, blow the small pieces by a pipette, collect the small pieces of pluripotent stem cells and transfer the small pieces into a new culture dish according to a certain proportion for amplification culture. The preparation of the product has extremely high requirements on equipment materials and processes, so that the cost is high, 10 sales prices in China are about 2500-3200 yuan, the product cannot be sterilized and can only be used once, and the cost is extremely incompatible with the basic research of most laboratories in China.
Disclosure of Invention
Therefore, the invention aims to provide a passage method of pluripotent stem cells, which solves the problems of high cost and poor stability of the existing passage method.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows: a method of passaging pluripotent stem cells, the method comprising the steps of:
s1, culturing pluripotent stem cells in a culture dish to obtain a pluripotent stem cell colony;
s2, removing the culture medium in the culture dish from the hES which reaches 70-80% fusion, and enabling the passage tool to move on the surface of the culture dish containing the culture medium, and sequentially pushing and rolling the passage tool from right to left;
s3, horizontally rotating the direction of the culture dish, and repeating the step S2 to obtain a cut pluripotent stem cell sheet;
s4, adding dissociation solution into the culture dish, and placing the dissociation solution into the culture dish at 36-38 ℃ and 5% CO 2 Incubating the incubator for 3 to 7 minutes to obtain the harvestA compressed pluripotent stem cell sheet;
s5, blowing the bottom of the culture dish, eluting the contracted pluripotent stem cell pieces, and transferring the pluripotent stem cell pieces into a centrifuge tube;
s6, adding the dissociation solution, and repeating the step S5 to obtain the eluted pluripotent stem cell sheet;
s7, centrifuging the eluted pluripotent stem cell slices, and removing supernatant to obtain centrifuged pluripotent stem cells;
s8, adding a new culture medium into the centrifuged pluripotent stem cells for resuspension, and then carrying out the steps of 1: and (4-6) inoculating the re-suspended pluripotent stem cells into a new culture dish according to the proportion, and thus completing the passage of the pluripotent stem cells.
Preferably, in the step S2, the passage tool is a spring cutter, the spring cutter includes a precision spring, a roller and tweezers, a clamping groove is provided on an outer side wall of the roller, and when in use, the precision spring is sleeved on the roller, and an end of the tweezers is embedded into the clamping groove on the outer side wall of the roller, so as to compress the precision spring and adjust the spacing between the precision springs.
Preferably, the diameter of the precision spring is 5-10 mm; the diameter of the spring wire forming the precision spring is 0.1-0.2 mm, and the length of the spring wire is 1-2 cm; the spacing between the spring wires is 0.1-0.2.
Preferably, the diameter of the roller is 4-8 mm, and the height is 1-2 cm; the number of the clamping grooves is at least two, the length, the width and the height of at least two clamping grooves are all 1-2 mm, and the spacing between the at least two clamping grooves is 0.2-0.8 cm.
Preferably, the forceps are flat-headed at the ends, and the thickness of the ends is 0.2-0.4 mm.
Preferably, in the step S3, the horizontal rotation is performed at an angle ranging from 85 ° to 95 °.
Preferably, in the step S4, the dissociation liquid is composed of 1 XDPBS to which 2500 to 3500mg/L of glucose is added.
Preferably, in the step S6, the volume of the dissociation liquid added is 2 to 7ml.
Preferably, in S7, the rotational speed at the time of centrifugation is 500 to 1000rpm.
Compared with the prior art, when the method is used, the pluripotent stem cell colony is cut by rotating the passage tool, and the pluripotent stem cell colony in the culture dish can be cut into square small pieces by repeatedly rolling and pushing the passage tool in a way of crossing at 85-95 degrees, then dissociation liquid is added, and the temperature is between 36 and 38 ℃ and the concentration of CO is 5 percent 2 The culture medium of the pluripotent stem cells can be effectively kept from being destroyed, and the activity of the pluripotent stem cells in passage is further improved because the dissociating liquid used in the process does not contain any dissociating enzyme or calcium ion chelating agent; in addition, the cut stem cell pieces shrink, the pluripotent stem cell pieces can be peeled off by blowing the culture medium, the suspension is collected and centrifuged to be resuspended, and the suspension is inoculated into a new culture dish to finish the passage of the pluripotent stem cells, so that the process not only realizes the purpose of simply and effectively cutting the pluripotent stem cell colony into the uniform-sized clusters, but also achieves the purposes of shortening the passage time and reducing the culture cost, thereby having great application value and popularization significance; in addition, the passage method is simple and easy to operate, and the method is widely popularized and used.
Drawings
FIG. 1 is a side view of a roller in a passage tool according to example 1 of the present invention;
FIG. 2 is a physical diagram of the cut pluripotent stem cell sheet obtained in example 1 of the invention;
FIG. 3 is a schematic structural view of a precision spring in a passage tool according to embodiment 1 of the present invention;
fig. 4 is a schematic perspective view of a roller in a passage tool according to example 1 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The starting materials used in the examples below were all available either directly or prepared by prior art techniques.
The embodiment of the invention provides a passage method of pluripotent stem cells, which is realized by the following steps:
s1, culturing pluripotent stem cells in a culture dish to obtain a pluripotent stem cell colony;
s2, removing the culture medium in the culture dish from the hES which reaches 70-80% fusion, and enabling the passage tool to move on the surface of the culture dish containing the culture medium, and sequentially pushing and rolling the passage tool from right to left;
s3, rotating the direction of the culture dish by 85-95 degrees horizontally, and repeating the procedure of S2 to obtain the multi-functional stem cell sheet to be cut;
s4, adding a dissociation solution composed of glucose of 2500-3500 mg/L added with 1 xDPBS into the culture dish, and placing the dissociation solution into the culture dish at 36-38 ℃ and 5% CO 2 Incubating the mixture in an incubator for 3 to 7 minutes to obtain a contracted pluripotent stem cell sheet;
s5, blowing the bottom of the culture dish by using a pipette of 5ml, eluting the contracted pluripotent stem cell pieces, and transferring the pluripotent stem cell pieces into a centrifuge tube of 15 ml;
s6, adding 2-7 ml of the dissociation liquid, and repeating the step S5 to obtain the eluted pluripotent stem cell sheet;
s7, centrifuging the eluted pluripotent stem cell slices under the condition of 500-1000 rpm, and removing supernatant to obtain centrifuged pluripotent stem cells;
s8, adding a new culture medium into the centrifuged pluripotent stem cells for resuspension, and then carrying out the steps of 1: and (4-6) inoculating the re-suspended pluripotent stem cells into a new culture dish according to the proportion, and thus completing the passage of the pluripotent stem cells.
In addition, in the step S2, the passage tool is a spring cutter, as shown in fig. 1-3, the spring cutter includes a precision spring 1, a roller 2, and tweezers (not shown in the drawings), a clamping groove 21 is provided on the outer side wall of the roller 2, and when in use, the precision spring 1 is sleeved on the roller 2, and the end of the tweezers is embedded into the clamping groove 21 on the outer side wall of the roller 2, so as to compress the precision spring 1 and adjust the spacing between the precision springs 1; the diameter of the precision spring 1 is 5-10 mm (namely, the distance denoted by 'D' in figure 1); the diameter of the spring wire constituting the precision spring 1 is 0.1 to 0.2mm (i.e., the distance denoted by "d" in fig. 1), and the length of the spring wire is 1 to 2cm, preferably 1.5cm; the spacing of the spring wires is 0.1 to 0.2, preferably 0.15mm (i.e. the distance denoted by "t" in fig. 1); the diameter of the roller 2 is 4-8 mm, and the height is 1-2 cm (namely, the distance denoted by 'H' in figure 1); the number of the clamping grooves 21 is at least two, the length, the width and the height of at least two clamping grooves 21 are all 1-2 mm, and the distance between at least two clamping grooves 21 is 0.2-0.8 cm; the end parts of the tweezers are flat heads, and the thickness of the end parts is 0.2-0.4 mm.
After the scheme is adopted, the pluripotent stem cell colony is cut by rotating the passage tool, the pluripotent stem cell colony in the culture dish can be cut into square small pieces corresponding to the space between the spring wires by rolling and pushing the passage tool which is crossed for a plurality of times at 85-95 degrees, then dissociation liquid is added, and the temperature is between 36 and 38 ℃ and the concentration of CO is 5 percent 2 The culture medium of the pluripotent stem cells can be effectively kept from being destroyed, and the activity of the pluripotent stem cells in passage is further improved because the dissociating liquid used in the process does not contain any dissociating enzyme or calcium ion chelating agent; in addition, the cut stem cell pieces shrink, the pluripotent stem cell pieces can be peeled off by flushing the culture medium, the suspension is collected and centrifuged to be resuspended, and the pluripotent stem cells can be transferred to a new culture dish after being inoculated, so that the aim of simply and effectively cutting pluripotent stem cell colonies into uniform-sized clusters is fulfilled, and the aims of shortening transfer time and reducing culture cost are fulfilled, so that the pluripotent stem cell pieces have great application value and popularization significance.
The following are specific examples
Example 1
The method for passaging pluripotent stem cells provided in example 1 is realized by the following steps:
s1, culturing pluripotent stem cells in a culture dish to obtain a pluripotent stem cell colony;
s2, removing the culture medium in the culture dish from the hES which reaches 70-80% fusion, and enabling the passage tool to move on the surface of the culture dish containing the culture medium, and sequentially pushing and rolling the passage tool from right to left;
the passage tool is a spring cutter, the spring cutter comprises a precise spring 1, a rolling shaft 2 and tweezers, a clamping groove 21 is formed in the outer side wall of the rolling shaft 2, when the passage tool is used, the precise spring 1 is sleeved on the rolling shaft 2, and the end parts of the tweezers are embedded into the clamping groove 21 in the outer side wall of the rolling shaft 2 and used for compressing the precise spring 1 and adjusting the spacing of the precise spring 1; the diameter of the precision spring 1 is 5-10 mm; the diameter of the spring wire constituting the precision spring 1 is 0.15mm, and the length of the spring wire is 1.5cm; the spacing between the spring wires is 0.15mm; the diameter of the roller 2 is 4-8 mm, and the height is 1.6cm; the number of the clamping grooves 21 is at least two, the length, the width and the height of at least two clamping grooves 21 are all 1.5mm, and the distance between at least two clamping grooves 21 is 0.5cm; the end parts of the tweezers are flat heads, and the thickness of the end parts is 0.2-0.4 mm;
s3, rotating the direction of the culture dish by 90 degrees horizontally, and repeating the procedure of S2 to obtain a pluripotent stem cell sheet to be cut;
s4, adding a dissociation solution composed of glucose added with 300mg/L of 1 xDPBS into the culture dish, and placing the dissociation solution into 5% CO at 37 DEG C 2 Incubating the culture box for 5min to obtain a contracted pluripotent stem cell sheet;
s5, blowing the bottom of the culture dish by using a pipette of 5ml, eluting the contracted pluripotent stem cell pieces, and transferring the pluripotent stem cell pieces into a centrifuge tube of 15 ml;
s6, adding 5ml of the dissociation liquid, and repeating the step S5 to obtain the eluted pluripotent stem cell sheet;
s7, centrifuging the eluted pluripotent stem cell slices under the condition that the rotating speed is 800rpm, and removing supernatant to obtain centrifuged pluripotent stem cells;
s8, adding a new culture medium into the centrifuged pluripotent stem cells for resuspension, and then carrying out the steps of 1: and (4-6) inoculating the re-suspended pluripotent stem cells into a new culture dish according to the proportion, and thus completing the passage of the pluripotent stem cells.
Example 2
The method for passaging pluripotent stem cells provided in example 2 is realized by the following steps:
s1, culturing pluripotent stem cells in a culture dish to obtain a pluripotent stem cell colony;
s2, removing the culture medium in the culture dish from the hES which reaches 70-80% fusion, and enabling the passage tool to move on the surface of the culture dish containing the culture medium, and sequentially pushing and rolling the passage tool from right to left; the passaging tool used in this step was the same as that in example 1;
s3, rotating the direction of the culture dish by 85 degrees horizontally, and repeating the procedure of S2 to obtain a pluripotent stem cell sheet to be cut;
s4, adding dissociation liquid composed of glucose with 2500mg/L added by 1 xDPBS into the culture dish, placing into 37 ℃ and 5% CO 2 Incubating the culture box for 3min to obtain a contracted pluripotent stem cell sheet;
s5, blowing the bottom of the culture dish by using a pipette of 5ml, eluting the contracted pluripotent stem cell pieces, and transferring the pluripotent stem cell pieces into a centrifuge tube of 15 ml;
s6, adding 2ml of the dissociation liquid, and repeating the step S5 to obtain the eluted pluripotent stem cell sheet;
s7, centrifuging the eluted pluripotent stem cell slices under the condition that the rotating speed is 500rpm, and removing supernatant to obtain centrifuged pluripotent stem cells;
s8, adding a new culture medium into the centrifuged pluripotent stem cells for resuspension, and then carrying out the steps of 1: and (4-6) inoculating the re-suspended pluripotent stem cells into a new culture dish according to the proportion, and thus completing the passage of the pluripotent stem cells.
Example 3
The method for passaging the pluripotent stem cells provided by the embodiment 3 of the invention is realized by the following steps:
s1, culturing pluripotent stem cells in a culture dish to obtain a pluripotent stem cell colony;
s2, removing the culture medium in the culture dish from the hES which reaches 70-80% fusion, and enabling the passage tool to move on the surface of the culture dish containing the culture medium, and sequentially pushing and rolling the passage tool from right to left; the passaging tool used in this step was the same as that in example 1;
s3, rotating the direction of the culture dish by 95 degrees horizontally, and repeating the procedure of S2 to obtain a pluripotent stem cell sheet to be cut;
s4, adding a dissociation solution composed of 3500mg/L glucose added with 1 xDPBS into the culture dish, and placing into 37 ℃ and 5% CO 2 Incubating the culture box for 7min to obtain a contracted pluripotent stem cell sheet;
s5, blowing the bottom of the culture dish by using a pipette of 5ml, eluting the contracted pluripotent stem cell pieces, and transferring the pluripotent stem cell pieces into a centrifuge tube of 15 ml;
s6, adding 7ml of the dissociation liquid, and repeating the step S5 to obtain the eluted pluripotent stem cell sheet;
s7, centrifuging the eluted pluripotent stem cell sheet under the condition of 1000rpm, and removing supernatant to obtain centrifuged pluripotent stem cells;
s8, adding a new culture medium into the centrifuged pluripotent stem cells for resuspension, and then carrying out the steps of 1: and (4-6) inoculating the re-suspended pluripotent stem cells into a new culture dish according to the proportion, and thus completing the passage of the pluripotent stem cells.
Example 4
The method for passaging the pluripotent stem cells provided by the embodiment 4 of the invention is realized by the following steps:
s1, culturing pluripotent stem cells in a culture dish to obtain a pluripotent stem cell colony;
s2, removing the culture medium in the culture dish from the hES which reaches 70-80% fusion, and enabling the passage tool to move on the surface of the culture dish containing the culture medium, and sequentially pushing and rolling the passage tool from right to left;
s3, rotating the direction of the culture dish by 90 degrees horizontally, and repeating the procedure of S2 to obtain a pluripotent stem cell sheet to be cut;
s4, adding a dissociation solution composed of 1 xDPBS added with 3000mg/L glucose into the culture dish, and placing the dissociation solution into 37 ℃ and 5% CO 2 Incubating the culture box for 7min to obtain a contracted pluripotent stem cell sheet;
s5, blowing the bottom of the culture dish by using a pipette of 5ml, eluting the contracted pluripotent stem cell pieces, and transferring the pluripotent stem cell pieces into a centrifuge tube of 15 ml;
s6, adding 7ml of the dissociation liquid, and repeating the step S5 to obtain the eluted pluripotent stem cell sheet;
s7, centrifuging the eluted pluripotent stem cell slices under the condition that the rotating speed is 800rpm, and removing supernatant to obtain centrifuged pluripotent stem cells;
s8, adding a new culture medium into the centrifuged pluripotent stem cells for resuspension, and then carrying out the steps of 1: and (4-6) inoculating the re-suspended pluripotent stem cells into a new culture dish according to the proportion, and thus completing the passage of the pluripotent stem cells.
Example 5
The method for passaging the pluripotent stem cells provided by the embodiment 5 of the invention is realized by the following steps:
s1, culturing pluripotent stem cells in a culture dish to obtain a pluripotent stem cell colony;
s2, removing the culture medium in the culture dish from the hES which reaches 70-80% fusion, and enabling the passage tool to move on the surface of the culture dish containing the culture medium, and sequentially pushing and rolling the passage tool from right to left;
s3, rotating the direction of the culture dish by 90 degrees horizontally, and repeating the procedure of S2 to obtain a pluripotent stem cell sheet to be cut;
s4, adding a dissociation solution composed of 1 xDPBS added with 3000mg/L glucose into the culture dish, and placing the dissociation solution into 37 ℃ and 5% CO 2 Incubating the culture box for 3min to obtain a contracted pluripotent stem cell sheet;
s5, blowing the bottom of the culture dish by using a pipette of 5ml, eluting the contracted pluripotent stem cell pieces, and transferring the pluripotent stem cell pieces into a centrifuge tube of 15 ml;
s6, adding 2ml of the dissociation liquid, and repeating the step S5 to obtain the eluted pluripotent stem cell sheet;
s7, centrifuging the eluted pluripotent stem cell sheet under the condition of 1000rpm, and removing supernatant to obtain centrifuged pluripotent stem cells;
s8, adding a new culture medium into the centrifuged pluripotent stem cells for resuspension, and then carrying out the steps of 1: and (4-6) inoculating the re-suspended pluripotent stem cells into a new culture dish according to the proportion, and thus completing the passage of the pluripotent stem cells.
In order to verify the effect of the passage tool consisting of the precision spring 1, the roller 2 and the tweezers, the cut pluripotent stem cell sheet obtained in step 3 of example 1 was first tested, the test results are shown in figure 1,
further knowledge can be obtained by observing fig. 1: cutting the pluripotent stem cell colony by rotating a passage tool, cutting the pluripotent stem cell colony in a culture dish into square small pieces corresponding to the space between spring wires by rolling and pushing the passage tool for multiple times at 85-95 degrees, adding dissociation liquid, and carrying out CO concentration at 36-38 DEG and 5% 2 The culture medium of the pluripotent stem cells can be effectively kept from being destroyed, and the activity of the pluripotent stem cells in passage is further improved because the dissociating liquid used in the process does not contain any dissociating enzyme or calcium ion chelating agent; in addition, the cut stem cell pieces can shrink, the culture medium is flushed to enable the pluripotent stem cell pieces to be peelable, the suspension is collected and centrifuged to be resuspended, and the culture medium is inoculated into a new culture dish to finish passage of pluripotent stem cells, the process not only achieves the purpose of simply and effectively cutting pluripotent stem cell colonies into clusters with uniform size, but also achieves the purpose of shortening passage time and reducing culture cost, in addition, according to the distance between the regulating spring wires, the size of the cut pluripotent stem cell pieces can be effectively controlled, the requirements of different pluripotent stem cell lines and researches are met, and the method has great application value and popularization significance.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.
Claims (7)
1. A method for passaging pluripotent stem cells, comprising the steps of:
s1, culturing pluripotent stem cells in a culture dish to obtain a pluripotent stem cell colony;
s2, removing the culture medium in the culture dish from the hES which reaches 70-80% fusion, and enabling the passage tool to move on the surface of the culture dish containing the culture medium, and sequentially pushing and rolling the passage tool from right to left;
the passage tool is a spring cutter which comprises a precise spring (1), a roller (2) and tweezers, wherein a clamping groove (21) is formed in the outer side wall of the roller (2), and when the passage tool is used, the precise spring (1) is sleeved on the roller (2) and the end part of the tweezers is embedded into the clamping groove (21) in the outer side wall of the roller (2) and used for compressing the precise spring (1) and adjusting the distance between the precise springs (1);
s3, horizontally rotating the direction of the culture dish, and repeating the step S2 to obtain a cut pluripotent stem cell sheet;
s4, adding dissociation liquid into the culture dish, and placing the dissociation liquid into the culture dish at 36-38 ℃ and 5% CO 2 Incubating the culture box for 3-7 min to obtain a contracted pluripotent stem cell sheet;
s5, blowing the bottom of the culture dish, eluting the contracted pluripotent stem cell pieces, and transferring the pluripotent stem cell pieces into a centrifuge tube;
s6, adding the dissociation solution, and repeating the step S5 to obtain the eluted pluripotent stem cell sheet;
s7, centrifuging the eluted pluripotent stem cell slices, and removing supernatant to obtain centrifuged pluripotent stem cells; the dissociation liquid is composed of 1 XDPBS added with 2500-2500 mg/L glucose;
s8, adding a new culture medium into the centrifuged pluripotent stem cells for resuspension, and then carrying out the steps of 1: and (4-6) inoculating the resuspended pluripotent stem cells into a new culture dish to finish the passage of the pluripotent stem cells.
2. The method for passaging pluripotent stem cells according to claim 1, wherein the diameter of the precision spring (1) is 5-10 mm; the diameter of a spring wire forming the precision spring (1) is 0.1-0.2 mm, and the length of the spring wire is 1-2 cm; the distance between the spring wires is 0.1-0.2.
3. The method for passaging pluripotent stem cells according to claim 2, wherein the diameter of the roller (2) is 4-8 mm and the height is 1-2 cm; the number of the clamping grooves (21) is at least two, the length, the width and the height of at least two clamping grooves (21) are all 1-2 mm, and the distance between at least two clamping grooves (21) is 0.2-0.8 cm.
4. The method for passaging pluripotent stem cells according to claim 3, wherein the forceps have flat ends and the thickness of the ends is 0.2-0.4 mm.
5. The method for passaging pluripotent stem cells according to any one of claims 1 to 4, wherein in S3, the horizontal rotation is in the range of 85 ° to 95 °.
6. The method for passaging pluripotent stem cells according to claim 5, wherein the volume of the dissociation liquid added in S6 is 2-7 ml.
7. The method for passaging pluripotent stem cells according to claim 6, wherein in S7, the rotational speed at the time of centrifugation is 500 to 1000rpm.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN201793578U (en) * | 2010-09-01 | 2011-04-13 | 湖南光琇高新生命科技有限公司 | Stem cell multifunctional combined passage tool |
| CN106381282A (en) * | 2016-10-12 | 2017-02-08 | 广东艾时代生物科技有限责任公司 | Induced pluripotent stem cell subculture method |
| CN110291189A (en) * | 2017-01-17 | 2019-09-27 | 隆萨沃克斯维尔股份有限公司 | The passage of unicellular people's pluripotent stem cell and harvest preparation |
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| US8828720B2 (en) * | 2008-03-17 | 2014-09-09 | Agency For Science, Technology And Research | Microcarriers for stem cell culture |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201793578U (en) * | 2010-09-01 | 2011-04-13 | 湖南光琇高新生命科技有限公司 | Stem cell multifunctional combined passage tool |
| CN106381282A (en) * | 2016-10-12 | 2017-02-08 | 广东艾时代生物科技有限责任公司 | Induced pluripotent stem cell subculture method |
| CN110291189A (en) * | 2017-01-17 | 2019-09-27 | 隆萨沃克斯维尔股份有限公司 | The passage of unicellular people's pluripotent stem cell and harvest preparation |
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