CN110564674B - Stem cell bottoming working solution and application thereof - Google Patents
Stem cell bottoming working solution and application thereof Download PDFInfo
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- CN110564674B CN110564674B CN201910935503.0A CN201910935503A CN110564674B CN 110564674 B CN110564674 B CN 110564674B CN 201910935503 A CN201910935503 A CN 201910935503A CN 110564674 B CN110564674 B CN 110564674B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Abstract
The invention belongs to the technical field of stem cell culture, and particularly relates to a stem cell bottoming working solution and application thereof. The invention provides stem cell bottom-paving working solution, which comprises the following components: matrigel and VTN-N; the volume ratio of the Matrigel to the VTN-N is 2: 1-3: 1. The experimental result shows that the stem cell bottoming working solution with the volume ratio of Matrigel to VTN-N of 2: 1-3: 1 is adopted for bottoming treatment and then stem cell recovery, so that the recovery effect is good, the aggregate growth can be reduced, the growth speed is accelerated, and the cell death is reduced; the stem cell bottom paving working solution is adopted for bottom paving treatment and then recovery culture, and the number growth of the stem cells accords with the growth trend of the stem cells.
Description
Technical Field
The invention belongs to the technical field of stem cell culture, and particularly relates to a stem cell bottoming working solution and application thereof.
Background
Embryonic Stem Cells (ESCs) can be differentiated into all cells with 3 germ layers in vivo, and then participate in forming all tissues and organs of an organism, so that the method has great application value in the fields of regenerative medicine, tissue engineering, drug discovery and evaluation and the like. However, the ethical, immunological rejection, resource shortage and other problems faced by ESCs hinder the development of ESCs in basic research and clinical application of stem cell biology, and the acquisition method of human induced pluripotent stem cells (hipscs) can well overcome the problems. Thus, in the field also referred to as "stem cells", hipscs avoid both physiological ethical issues and uncontrolled differentiation differences compared to embryonic stem cells.
The hiPSC is a type of pluripotent stem cells with the molecular characteristics, the renewal capacity and the differentiation potential similar to those of embryonic early pluripotent stem cells, can be directionally differentiated into specific functional cells, tissues and organs through artificial intervention, and can be successfully differentiated into neuron cells, glial cells, cardiovascular cells, primordial germ cells and the like in vitro, thereby providing a brand-new way for disease mechanism research and cell therapy. The prior research shows that Oct4 gene and Nanog gene jointly regulate the growth and development of cells and maintain totipotency through hipSC, and Lin gene and Fgf4 gene play an important role in maintaining totipotency of cells and promote cell proliferation and tissue repair.
However, the environmental requirements of the in vitro resuscitation and culture of hipscs are strict, and the conventional culture mode for resuscitation or culture has the problems of easy aggregation growth, slow growth or high death rate.
Disclosure of Invention
In view of the above, the invention provides a stem cell bottom-paving working solution and an application thereof, which are used for solving the problems of easy aggregation growth, slow growth or high death rate of resuscitation or culture stem cells in the prior art.
The specific technical scheme of the invention is as follows:
a stem cell bottoming working fluid, comprising: matrigel and VTN-N;
the volume ratio of the Matrigel to the VTN-N is 2: 1-3: 1.
The experimental result shows that the stem cell bottoming working solution with the volume ratio of Matrigel to VTN-N of 2: 1-3: 1 is adopted for bottoming treatment and then stem cell recovery, so that the recovery effect is good, the aggregate growth can be reduced, the growth speed is accelerated, and the cell death is reduced; the stem cell bottom paving working solution is adopted for bottom paving treatment and then recovery culture, and the number growth of the stem cells accords with the growth trend of the stem cells.
Preferably, the volume ratio of the Matrigel to the VTN-N is 3: 1.
Preferably, the volume ratio of the Matrigel to the VTN-N is 2: 1.
Preferably, the concentration of the Matrigel in the stem cell bottom-paving working solution is 6.67-7.5 muL/mL;
the concentration of the VTN-N in the stem cell bottom-paving working solution is 2.5-3.33 mu L/mL.
In the invention, the preparation method of the stem cell bottom-paving working solution comprises the following steps: and respectively freezing and thawing the Matrigel and the VTN-N, diluting by adopting DPBS to obtain a Matrigel diluent and a VTN-N diluent, and mixing the Matrigel diluent and the VTN-N diluent to obtain a stem cell bottoming working solution.
The invention also provides application of the stem cell bottoming working solution in the technical scheme in stem cell recovery.
The invention also provides a method for resuscitating stem cells, which comprises the following steps:
and paving the bottom of the culture container by using the stem cell bottom paving working solution, discarding the stem cell bottom paving working solution after paving the bottom, and inoculating stem cells into the culture container for resuscitation.
Preferably, the stem cell is iPSC, PSC or ESC, more preferably hiPSC, and even more preferably hiPSC-U1.
Preferably, the bottom paving specifically comprises:
adding the stem cell bottom-laying working solution into a culture container, and then placing at 37 ℃ and 5% CO2The culture box is incubated for 12 to 24 hours, and more preferably for 24 hours.
The invention also provides application of the stem cell bottom-paving working solution in stem cell culture.
Preferably, the method comprises the following steps:
and paving the bottom of the culture container by using the stem cell bottom paving working solution, discarding the stem cell bottom paving working solution after paving the bottom, and inoculating stem cells into the culture container for cell culture.
In summary, the present invention provides a stem cell bottom-plating working solution, including: matrigel and VTN-N; the volume ratio of the Matrigel to the VTN-N is 2: 1-3: 1. The experimental result shows that the stem cell bottoming working solution with the volume ratio of Matrigel to VTN-N of 2: 1-3: 1 is adopted for bottoming treatment and then stem cell recovery, so that the recovery effect is good, the aggregate growth can be reduced, the growth speed is accelerated, and the cell death is reduced; the stem cell bottom paving working solution is adopted for bottom paving treatment and then recovery culture, and the number growth of the stem cells accords with the growth trend of the stem cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a 10X optical microscope photograph of group 1 cells after 24 hours of resuscitation in example 1 of the present invention;
FIG. 2 is a 10X optical microscope photograph of group 2 cells after 24 hours of resuscitation in example 1 of the present invention;
FIG. 3 is a 10X optical microscope photograph of group 3 cells after 24 hours of resuscitation in example 1 of the present invention;
FIG. 4 is a 10X optical microscope photograph of group 4 cells after 24 hours of resuscitation in example 1 of the present invention;
FIG. 5 is a 10X optical microscope photograph of group 5 cells of example 1 after 24 hours of resuscitation;
FIG. 6 is a 10X optical microscope photograph of group 6 cells after 24 hours of resuscitation in example 1 of the present invention;
FIG. 7 is a 10X optical microscope photograph of group 7 cells after 24 hours of resuscitation in example 1 of the present invention;
FIG. 8 is a graph of the growth numbers of the resuscitative cultures after the bottoming treatment with the group 5 stem cell bottoming working solution in example 2 of the present invention;
in fig. 1 to 7, the wavy circle marks indicate cells with better growth, the circles indicate aggregated cells, and the arrows indicate dead cells.
Detailed Description
The invention provides a stem cell bottoming working solution and application thereof, which are used for solving the problems of easy aggregation growth, slow growth or high death rate of resuscitation or stem cell culture in the prior art.
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In a specific embodiment of the invention, Matrigel was purchased from CORNING, lot number 354277; VTN-N was purchased from Gibco, lot A14700; DPBS was purchased from GEN-VIEW, lot number GP 3302; the hiPSC is specifically hiPSC-U1, available from beijing seebeck co ltd, CA 4002106; TeSR-E8 medium was purchased from Stem Cell, cat.05990; y-27632 was purchased from TOCRIS, Cat.1254.
Example 1
1) Preparing stem cell bottoming working solution of different groups
The Matrigel and VTN-N are put into a4 ℃ overnight freeze-thaw chamber from-80 ℃, 100 mu L of the Matrigel and VTN-N are respectively added into a 10mL DPBS to be diluted in a 15mL centrifuge tube according to the proportion of 1:100 on the next day, and Matrigel diluent and VTN-N diluent are obtained and stored in a4 ℃ refrigerator for standby.
Different groups of stem cell bottom-paving working solutions are prepared by adopting Matrigel diluent and VTN-N diluent, the total volume of the stem cell bottom-paving working solutions is 2mL, and the method comprises the following specific steps:
group 1: 2mL of Matrigel diluent;
group 2: 2mL of VTN-N diluent;
group 3: 1mL of Matrigel diluent and 1mL of VTN-N diluent;
group 4: 1.33mL of Matrigel diluent and 0.67mL of VTN-N diluent;
group 5: 1.5mL of Matrigel diluent and 0.5mL of VTN-N diluent;
group 6: 0.67mL of Matrigel diluent and 1.33mL of VTN-N diluent;
group 7: matrigel dilution 0.5mL + VTN-N dilution 1.5 mL.
2) Bottoming culture dish
According to the grouping, 2mL of stem cell plating solution was added to 7 groups of 60mm culture dishes, and 5% CO was added at 37 ℃%2Incubate for 24 h.
3) Thawing treatment of hiPSC
The frozen tube containing the cells (hiPSC-U1) was taken out from a liquid nitrogen tank at-196 ℃ and the cell density of each tube was about 10000 cells/ml, and the cells were thawed by gently and rapidly shaking in a thermostatic water bath at 37 ℃ for about 1 min. Then the water on the freezing storage tube is wiped clean by absorbent paper, and then alcohol is sprayed on the freezing storage tube, and finally the freezing storage tube is placed in a super clean bench.
4) Centrifugal precipitation of cell suspensions
After the aggregated cells were blown off by gently tapping the cell suspension 1 to 3 times using a pipette gun, the cell suspension was transferred into a 15ml centrifuge tube containing 9ml of TeSR-E8 medium, gently tapped for 1 time, and then placed in a centrifuge and centrifuged at 1000r/min at room temperature for 4 min.
5) Resuspending the pelleted cells after centrifugation into a cell suspension
Quickly pouring out the solution in the centrifugal tube after centrifugation in the step 4), if residual solution exists in the centrifugal tube, quickly sucking and discarding the residual solution by using a pipette gun to leave deposited cells, adding 4ml of culture solution, slightly blowing and beating the cell suspension for 5 times, and repeating the operation for 7 groups to prepare seven groups of cell concentration suspensions. Wherein the culture medium is prepared by adding 1 mu L Y-27632 of culture medium to 1ml of LTeSR-E8.
6) Resuscitating cells in culture dish
Sucking and discarding stem cell bottom-paving working solution in the 60mm culture dish after bottom-paving treatment in the step 2) by using a liquid-transferring gun, slightly blowing 7 groups of 4ml cell suspension prepared in the centrifugal tube in the step 5) for 1 time, respectively inoculating the cell suspension into 7 groups of 60mm culture dishes, quickly shaking the cell suspension back and forth and left and right, and placing the cell suspension at 37 ℃ and 5% CO2And (5) recovering for 24h in the incubator, and taking a picture by using an optical microscope.
Referring to fig. 1 to 7, the results of each group after 24h recovery show that the cells in group 1 grow in a small amount in an aggregation manner and grow faster; the cells of group 2 grew in a large amount of aggregates and grew slowly; the cells in the group 3 grow in a small amount in an aggregation manner, the growth is faster, and more dead cells exist; the cells of group 4 almost grow without aggregation and grow fast; the cells in the group 5 almost grow without agglomeration, the growth is fast, and the nuclear substance observation is obvious; the group 6 cells grow in a small amount in an aggregation manner, the growth is faster, and more dead cells exist; the cells in the group 7 grow more in an aggregated manner, the growth is faster, more dead cells are generated, and the results show that the recovery effect is good, the aggregated growth can be reduced, the growth speed is accelerated, and the cell death is reduced by adopting the stem cell bottom paving working solution with the volume ratio of Matrigel to VTN-N of 2: 1-3: 1 to perform bottom paving treatment and then perform stem cell recovery.
Example 2
The three groups of hipscs are subjected to bottoming treatment by using the group 5 stem cell bottoming working solution, then are recovered and cultured by the method in the embodiment 1, and the number of the hipscs in three days is counted, and the result is shown in fig. 8, wherein fig. 8 is a growth number diagram of the bottoming treatment and the recovery culture of the hipscs in the embodiment 2 by using the group 5 stem cell bottoming working solution, and the result shows that the number growth of the hipscs accords with the growth trend of stem cells when the bottoming treatment and the recovery culture are performed by using the stem cell bottoming working solution, and the stem cell bottoming working solution has effectiveness and feasibility.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A stem cell bottoming working solution is characterized by comprising: matrigel and VTN-N;
the volume ratio of the Matrigel to the VTN-N is 2: 1-3: 1.
2. The stem cell plating solution of claim 1, wherein the Matrigel and the VTN-N are in a volume ratio of 3: 1.
3. The stem cell plating solution of claim 1, wherein the Matrigel and the VTN-N are in a volume ratio of 2: 1.
4. The stem cell plating working solution of claim 1, wherein the concentration of Matrigel in the stem cell plating working solution is 6.67-7.5 μ L/mL;
the concentration of the VTN-N in the stem cell bottom-paving working solution is 2.5-3.33 mu L/mL.
5. Use of the stem cell bottoming working solution of any one of claims 1 to 4 for inducing pluripotent stem cell resuscitation in a human.
6. A method of resuscitating stem cells, comprising the steps of:
the stem cell bottoming working solution of any one of claims 1 to 4 is adopted to bottom a culture container, the stem cell bottoming working solution is discarded after bottoming is finished, and then human induced pluripotent stem cells are inoculated into the culture container for resuscitation.
7. The method according to claim 6, wherein the bottoming block comprises:
adding the stem cell bottom-laying working solution into a culture container, and then placing at 37 ℃ and 5% CO2Incubating for 12-24 h in the incubator.
8. Use of the stem cell bottoming working solution of any one of claims 1 to 4 in human induced pluripotent stem cell culture.
9. Use according to claim 8, characterized in that it comprises the following steps:
and paving the bottom of the culture container by using the stem cell bottom paving working solution, discarding the stem cell bottom paving working solution after paving the bottom, and inoculating human induced multifunctional stem cells into the culture container for cell culture.
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