CN113462639B

CN113462639B - Method for separating fibroblasts from human induced pluripotent stem cells and application of method

Info

Publication number: CN113462639B
Application number: CN202110922734.5A
Authority: CN
Inventors: 吴理达; 顾雨春
Original assignee: Chengnuo Regenerative Medical Technology Zhuhai Hengqin New Area Co ltd
Current assignee: Chengnuo Regenerative Medical Technology Zhuhai Hengqin New Area Co ltd
Priority date: 2021-08-12
Filing date: 2021-08-12
Publication date: 2022-05-27
Anticipated expiration: 2041-08-12
Also published as: WO2023016029A1; CN113462639A

Abstract

Compared with the prior art, the method provided by the invention has the advantages of short separation time, high separation efficiency, higher safety of the prepared fibroblasts, no need of forming embryoid bodies and replacing culture solution components for many times, capability of obtaining a large amount of high-purity fibroblasts suitable for clinical use in a short time, and very good clinical application prospect.

Description

Method for separating fibroblasts from human induced pluripotent stem cells and application of method

Technical Field

The invention belongs to the field of cell engineering, and particularly relates to a method for separating fibroblasts from human induced pluripotent stem cells and application thereof.

Background

Fibroblast (FB) is the most important cell in the dermal network layer of the skin, also called Fibroblast, and is a cell in the form of spindle or flat star existing in the honeycomb tissue or fibrous connective tissue, which can secrete structural proteins constituting the extracellular matrix, synthesize and secrete a large amount of collagen, and play an important role in maintaining the elasticity and toughness of the skin. The fibroblasts separated from the tissues have high proliferation potential and are widely applied to the aspects of skin burn repair, cosmetology, wound healing, regenerative medicine, biological safety detection, drug screening and the like.

Human induced pluripotent stem cells (ipscs) are pluripotent stem cells induced from Human terminally differentiated cells by an in vitro reprogramming technique, have in vitro self-renewal stability, and maintain normal karyotype and developmental pluripotency. Human iPSC or human cells, tissues or organs differentiated from the human iPSC are utilized to test the toxicological characteristics or drug effects of various biomedical materials, foods and medicines on human bodies, and the human iPSC can reflect the real conditions of the human bodies better than the human iPSC by using animal or immortalized cells. Therefore, the human iPSC and the differentiated cells thereof are expected to be developed into a brand-new biological safety detection and drug screening model. Fibroblasts obtained by differentiation of ipscs have sufficient cell sources, strong cell proliferation capacity, vigorous cell activity, and may have better effects of injury repair and aging resistance after reinfusion.

At present, the method for differentiating the iPSC into the fibroblast is mainly to induce differentiation through an embryo mimic (EB) approach, that is, the iPSC is aggregated into a spherical structure through a suspension drop culture or suspension culture technology, the structure is called an embryo mimic, in the differentiation process of the embryo mimic, more kinds of cell factors are often required to be added, and after a plurality of days of suspension drop culture or suspension culture, the cell is transferred into adherent culture, and then part of the cell is migrated into the fiber-like cell. The induced differentiation method of the embryoid body has the defects of low differentiation efficiency, complex differentiation process, long time consumption, more kinds of cell factors and higher cost. Therefore, the existing technology for differentiating iPSC into fibroblast has the problems of long time consumption, low yield, high technical requirement, limited cell purity and the like, restricts the large-scale culture of fibroblast, and limits the industrialization and marketization requirements of the fibroblast.

In view of the above, the present application provides a technology for directly separating fibroblasts from iPSC spontaneous differentiation products, which utilizes the phenomenon of spontaneous differentiation of an iPSC cell line in a certain proportion, wherein a lot of spontaneously differentiated cells are fibroblast-like cells, and can directly separate the spontaneously differentiated fibroblast-like cells from a culture by using a differential digestion method, so that the separated fibroblasts have the basic morphological characteristics and biological characteristics of the fibroblasts, can be expanded and cultured in vitro, further provide a technical basis for large-scale culture of autologous fibroblasts in the future, and can be used for beauty treatment, plastic treatment, wound healing, burn treatment, biological safety detection, drug screening and the like.

Disclosure of Invention

In order to overcome the technical defects in the prior art, the invention provides a method for separating fibroblasts derived from human induced pluripotent stem cells, which does not need to add cytokines or small molecular compounds, has high safety, short separation time and high separation efficiency, does not need animal-derived cells as a feeder layer in the separation process, does not need to form embryoid bodies, does not need to replace culture solution components for many times, and can obtain a large amount of high-purity fibroblasts suitable for clinical application in a short time.

The above object of the present invention is achieved by the following technical solutions:

in a first aspect of the invention, a method of isolating fibroblasts from induced pluripotent stem cells is provided.

Further, the method comprises the steps of:

(1) preparing induced pluripotent stem cells having spontaneously differentiated cells;

(2) removing induced pluripotent-like stem cells from the induced pluripotent stem cells with the spontaneously differentiated cells obtained in the step (1) to obtain spontaneously differentiated cells from which the induced pluripotent-like stem cells are removed;

(3) and (3) purifying the fibroblasts in the spontaneously differentiated cells of the induced pluripotent stem cells obtained in the step (2) to obtain purified fibroblasts.

The induced pluripotent stem cell, like the iPSC or the iPS cell, in the present invention refers to a pluripotent stem cell induced from a human terminally differentiated cell by an in vitro reprogramming technique, having in vitro self-renewal stability and maintaining normal karyotype and developmental pluripotency.

Further, the preparation of the induced pluripotent stem cells with spontaneously differentiated cells in the step (1) comprises:

(a) adding a digestive juice into the induced pluripotent stem cells, culturing until cells in the central area of the induced pluripotent stem cell colony start floating, and adding a culture medium to stop digestion after most of the cells fall off in a block shape;

(b) centrifuging the cell solution obtained in the step (a), removing supernatant, adding a culture medium and a ROCK inhibitor, and re-suspending cell precipitates to obtain cell suspension;

(c) placing the cell suspension obtained in the step (b) in a culture medium and a ROCK inhibitor, adjusting the cell density, culturing until the cell polymerization degree is increased to 75-85% or the diameter of any single cell colony is larger than the 10X microscopic field, and carrying out passage until at least 5% of spontaneously differentiated cells appear;

preferably, the digestive juice in step (a) comprises an accutase digestive juice, an EDTA digestive juice, a trypsin-EDTA digestive juice, a collagenase digestive juice;

more preferably, the digestive juice is accutase digestive juice;

most preferably, the accutase digest is an accutase digest preheated at 37 ℃;

most preferably, the amount of the accutase digestive juice is 1 mL;

preferably, the culturing conditions in step (a) are 37 ℃ and 5% CO₂；

Preferably, the incubation time in step (a) is 2-3 min;

preferably, the medium in step (a) is DMEM/F12 medium;

more preferably, the DMEM/F12 medium is pre-warmed DMEM/F12 medium at 37 ℃;

most preferably, the amount of DMEM/F12 medium is 5 mL;

preferably, the centrifugation in step (b) is performed at 200g for 5 min;

preferably, the medium in step (b) is E8 medium;

more preferably, the E8 medium is E8 medium pre-warmed at 37 ℃;

preferably, the ROCK inhibitors described in step (b) include Y27632, GSK429286A, RKI-1447, Y-33075dihydrochloride, Thiazoviin, K-115, SLx-2119, Chroman1, SAR407899, SR-3677;

more preferably, the ROCK inhibitor in step (b) is Y27632;

most preferably, the amount of the E8 medium in step (b) is 600-700. mu.L;

most preferably, the amount of Y27632 used in step (b) is 10. mu.M;

preferably, the medium in step (c) is E6 medium;

more preferably, the E6 medium is E6 medium pre-warmed at 37 ℃;

preferably, the ROCK inhibitors described in step (c) include Y27632, GSK429286A, RKI-1447, Y-33075dihydrochloride, Thiazoviin, K-115, SLx-2119, Chroman1, SAR407899, SR-3677;

more preferably, the ROCK inhibitor in step (c) is Y27632;

most preferably, the amount of E6 medium used in step (c) is 3 mL;

most preferably, the amount of Y27632 used in step (c) is 10. mu.M;

preferably, the cell density in step (c) is 1.2X 10⁴/cm²；

Preferably, the culturing conditions in step (c) are 37 ℃ and 5% CO₂；

Preferably, the culturing in step (c) further comprises repeating the changing of the medium every day;

preferably, the number of passages described in step (c) is 2 to 3.

Further, the induced pluripotent stem cells having spontaneously differentiated cells are at least 5% spontaneously differentiated cells present around the induced pluripotent stem cell colony, and the spontaneously differentiated cells are in the form of a long spindle;

preferably, the induced pluripotent stem cell having a spontaneously differentiated cell is derived from a mammal;

more preferably, the induced pluripotent stem cell having a spontaneously differentiated cell is derived from a human, mouse, rat, goat, sheep, pig, cat, rabbit, dog, wolf, horse, or cow;

most preferably, the induced pluripotent stem cells with spontaneously differentiated cells are of human origin.

Further, the removing process in the step (2) comprises: adding digestive juice into induced pluripotent stem cells with spontaneously differentiated cells, digesting and incubating until spontaneously differentiated cells exist around induced pluripotent stem cell colonies in a digestion product and obvious boundaries exist between the spontaneously differentiated cells and the induced pluripotent stem cell colonies, terminating digestion, removing the cells which can drop out from the digestion product, and collecting the spontaneously differentiated cells from which the induced pluripotent stem cells are removed;

preferably, the digestive juice comprises EDTA digestive juice, accutase digestive juice, trypsin-EDTA digestive juice, collagenase digestive juice;

more preferably, the digestive juice is EDTA digestive juice;

most preferably, the amount of the EDTA digestive solution is 0.5mM and 2 mL;

preferably, the time of digestion is 3-5 min;

preferably, the terminating digestion comprises discarding the digestion solution, and adding a culture medium to the cells after the digestion treatment;

more preferably, the medium is E8 complete medium;

preferably, the conditions for collecting the spontaneously differentiated cells from which the induced pluripotent stem cells are removed are 500-1000 rpm and centrifugation for 5 min;

more preferably, the conditions for collecting the spontaneously differentiated cells from which the induced pluripotent stem cells are removed are 800 rpm and centrifugation is performed for 5 min;

preferably, the digestion is preceded by washing the cells to be digested with a washing buffer;

more preferably, the washing buffer comprises DPBS buffer, PBS buffer, TBS buffer, PE buffer, D-Hanks balanced salt buffer, Earle's balanced salt buffer;

most preferably, the wash buffer is a DPBS buffer;

most preferably, the wash is a DPBS buffer wash 2 times for 1min each.

Further, the purification process in step (3) comprises: adding a digestive juice into the spontaneous differentiated cells from which the induced pluripotent stem cells are removed, digesting and incubating until the fibroblast-like cells become round and begin to fall off, stopping digestion, and collecting purified fibroblasts;

preferably, the digestive juice comprises accutase digestive juice, EDTA digestive juice, trypsin-EDTA digestive juice, collagenase digestive juice;

more preferably, the digestive juice is accutase digestive juice;

most preferably, the amount of the accutase digest is 4-fold dilution, 2 mL;

preferably, the time of digestion is 4-7 min;

more preferably, the incubation conditions are 37 ℃, 6 min;

more preferably, the medium is a high-glucose DMEM medium;

most preferably, the medium is a high-glucose DMEM medium containing 10% FBS;

preferably, the collection and purification conditions of the fibroblasts are 500-1000 rpm and 5min of centrifugation;

more preferably, the collecting condition of the purified fibroblasts is 800 r/min and 5min of centrifugation;

most preferably, the wash buffer is a DPBS buffer;

most preferably, the wash is a DPBS buffer wash 2 times for 1min each.

Further, the method further comprises removing epithelioid cells from the spontaneously differentiated cells;

preferably, said removing epithelioid cells from spontaneously differentiated cells comprises: adding digestive juice into the spontaneous differentiated cells from which induced pluripotent stem cells are removed, digesting and incubating until fibroblast-like cells become round and begin to fall off, and the shape of the epithelial-like cells is basically kept unchanged, and collecting the spontaneous differentiated cells from which the epithelial-like cells are removed;

more preferably, the digestive juice is accutase digestive juice;

most preferably, the amount of the accutase digest is 4-fold dilution, 2 mL;

preferably, the time of digestion is 4-7 min;

more preferably, the incubation conditions are 37 ℃, 6 min;

more preferably, the medium is a high-glucose DMEM medium;

most preferably, the medium is a high-glucose DMEM medium containing 10% FBS;

preferably, the collecting condition of the purified fibroblasts is 500-;

most preferably, the wash buffer is a DPBS buffer;

most preferably, the wash is a DPBS buffer wash 2 times for 1min each.

In a second aspect of the invention, there is provided a fibroblast cell or population of cells.

Further, the fibroblast cell or cell population is prepared by the method of the first aspect of the invention.

Further, cells obtained by further inducing differentiation or further reprogramming of the fibroblast or cell population prepared by the method of the present invention are also within the scope of the present invention.

In a third aspect of the invention, a feeder cell for culturing mature and commercial cells is provided.

Further, the feeder cells comprise fibroblasts or cell populations according to the second aspect of the invention.

In a fourth aspect of the invention, a cell therapy product is provided.

Further, the cell therapy product comprises the fibroblast cell or cell population according to the second aspect of the invention.

A fifth aspect of the invention provides the use of any one of the following:

(1) use of a fibroblast cell or cell population according to the second aspect of the invention in the manufacture of a cell product for the treatment, repair and/or prevention of a degenerative disc disease, heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated cardiomyopathy, ureteral reflux disease, gastroesophageal reflux disease, urinary incontinence, skin scars, skin ulcers, skin burns, large skin wounds, wrinkles, skin stretch marks, non-traumatic skin depressions, scars;

(2) use of a feeder cell according to the third aspect of the invention in the preparation of a mature and commercial cell;

(3) the cell therapy product according to the fourth aspect of the invention is used for the treatment, repair and/or prevention of degenerative disc disease, heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated cardiomyopathy, ureteral reflux disease, gastroesophageal reflux disease, urinary incontinence, skin scars, skin ulcers, skin burns, large skin wounds, wrinkles, skin expansion lines, non-traumatic skin depressions, scars.

In addition, the present invention provides a method of treating, repairing and/or preventing degenerative disc disease, heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated cardiomyopathy, ureteral reflux disease, gastroesophageal reflux disease, urinary incontinence, skin scars, skin ulcers, skin burns, large skin wounds, wrinkles, skin expansion lines, non-traumatic skin depressions, scars.

Further, the method comprises providing to a subject in need thereof an effective amount of the cell therapy product of the fourth aspect of the invention or the fibroblast cell or cell population of the second aspect of the invention.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) in the traditional embryoid body induced differentiation method, more cytokines or micromolecule compounds need to be added in the process of inducing iPSCs to differentiate into fibroblasts, and the use of more cytokines or micromolecule compounds leads to poor safety of the obtained fibroblasts and has potential carcinogenic risk; the method provided by the invention does not need to add cytokines or small molecular compounds, so that the safety of the fibroblasts prepared by the method is higher;

(2) in the traditional embryoid body induction differentiation method, in the process of inducing iPSC to differentiate into fibroblasts, animal-derived cells are required to be used as a feeder layer, embryoid bodies are formed, and meanwhile, culture solution components are required to be replaced for multiple times in the induction differentiation stage, so that the experimental operation is complex, the induction differentiation time is long, and the efficiency is low; the method provided by the invention has the advantages of short separation time and high separation efficiency, does not need animal source cells as a feeder layer in the separation process, does not need to form an embryoid body, does not need to replace culture solution components for many times, and can obtain a large amount of high-purity fibroblasts suitable for clinical use in a short time.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1 shows a graph of the results of spontaneous differentiation of ipscs, in which, panel a: results plot under one field, panel B: a result graph under another field of view;

FIG. 2 is a graph (10X) showing the results of morphology of iPSC colonies and surrounding spontaneously differentiated cells after sufficient incubation with DPBS;

figure 3 shows a graph of the results of iPSC colonies and spontaneously differentiated cell morphology after EDTA digestion for 5min (40 ×);

FIG. 4 is a graph showing the results of removing the morphology of spontaneously differentiated cells of iPSC;

FIG. 5 is a graph showing the results of fibroblast-like cell and epithelial-like cell morphology after 6 minutes of accutase digestion (200X);

FIG. 6 shows a graph of the results of purified fibroblast-like cells (100X);

FIG. 7 is a graph showing the results of reverse transcription PCR detection of iPSC spontaneously differentiating into fiber-like cells;

fig. 8 is a graph showing the results of immunofluorescence detection of ipscs spontaneously differentiating into fiber-like cells, wherein, a graph: VIMENTIN, panel B: DAPI, panel C: MERGE;

fig. 9 shows a result chart of flow identification of a cell surface marker in which ipscs spontaneously differentiate into fiber-like cells, in which, a chart: CD34, panel B: CD73, panel C: CD 105.

Detailed Description

The invention will now be further illustrated with reference to specific examples, which are provided for illustration only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The experimental methods not specifically mentioned in the following examples generally carry out the detection under the conventional conditions or according to the conditions recommended by the manufacturers, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 isolation of fibroblasts derived from iPSC

1. Experimental Material

(1) The information on the main reagents used in the examples of the present invention is shown in table 1;

table 1 information on the main reagents used in the examples of the present invention

(2) Formulation and preservation of reagents in the examples of the invention

High-glucose DMEM with 10% FBS: in a 50mL centrifuge tube, 40mL of high-sugar DMEM was aspirated, 10mL of FBS was added thereto, and the mixture was gently mixed and stored at 4 ℃ for 2 weeks.

2. Preparation of iPSC with spontaneously differentiated cells

The iPSC cell line (beijing, nori medicine science and technology ltd) used in this example is iPSC derived from PBMC reprogramming, and when the iPSC cell line is established, a certain proportion of spontaneously differentiated cells exist, are located around the iPSC colony, and have a long spindle shape, and the specific experimental method for preparing iPSC with spontaneously differentiated cells is as follows:

(1) passaging begins when iPSC cells expand to 75-85% degree of polymerization or either individual colony diameter is greater than 10X microscopic field. Taking a 6cm petri dish as an example, old medium was aspirated, washed twice with room temperature PBS, and then 1mL of 37The preheated Accutase is placed at 37 ℃ and 5% CO₂Observing the cell shedding degree under a microscope for 2-3min in a cell culture box until cells in a colony central area start floating;

(2) slightly vibrating the bottom of the culture dish, adding 5mL of DMEM/F12 Medium preheated at 37 ℃ after most of cells fall off in a block shape to neutralize the enzymolysis of Accutase, slightly blowing off the fallen cell blocks to small cell blocks by using a liquid transfer gun, and then transferring the small cell blocks to a 15mL centrifuge tube;

(3) centrifuging at room temperature for 5min at 200 g; removing the supernatant by using a pipette, adding 600-700 mu L of E8 culture medium preheated at 37 ℃ plus 10 mu M Y27632, and gently blowing and beating the resuspended cell sediment;

(4) from 37 ℃ with 5% CO₂The prepared Matrigel-coated cell culture plate was removed from the cell incubator, the liquid was removed, 3mL of 37 ℃ pre-warmed E6 medium + 10. mu. M Y27632 was added, followed by slow addition of 100. mu.L cell resuspension (i.e., 1: 6 or 1: 7 passages, optionally adjusted to the experimental requirements), at a final density of about 1.2X 10⁴cells/cm²；

(5) The cell plate was placed at 37 ℃ in 5% CO₂The cell culture box ensures that the cells are uniformly distributed on the surface of the culture plate as much as possible, and then stands overnight;

(6) replacing fresh E6 culture medium preheated at 37 ℃ after one day, repeating liquid replacement every day until the cell polymerization degree is increased to 75-85% or any single colony diameter is larger than 10X microscope field, and then performing passage operation;

(7) passage was repeated 2-3 times until at least 5% of differentiated cells appeared.

3. Culture of iPSC cells

(1) Experimental method

After iPSCs with spontaneously differentiated cells are cultured in a T25 culture flask (the used culture medium is E8 culture medium in Table 1) for 4-5 days, a certain proportion of spontaneously differentiated cells exist in the iPSCs, are positioned around iPSC colonies, and are in a long spindle shape;

(2) results of the experiment

The selected ipscs and the spontaneous differentiation states are shown in fig. 1A and fig. 1B, and it can be seen that the spontaneous differentiated cells are located around the iPSC colony, the shape is long spindle, and the iPSC colony is obvious and has an obvious boundary with the surrounding spontaneous differentiated cells.

4. iPS-like cells from iPSC depleted of spontaneously differentiated cells

(1) Experimental methods

When the iPSC colony in the cultured cell is obvious, spontaneous differentiated cells exist around the iPSC colony, and the iPSC colony and the peripheral spontaneous differentiated cells have obvious boundaries, removing the iPSC-like cells in the cultured cell, wherein the removing method comprises the following steps:

washing: washing the cultured cells twice by using DPBS (double stranded polymerase chain reaction), wherein each time is 1min, so that the cells at the edge of the iPSC colony begin to become round and begin to be separated from the surrounding spontaneous differentiated cells;

when the cells are washed twice, the cells are required to be washed sufficiently, the culture medium residue on the vertical wall of the culture bottle is noticed, and after the DPBS is washed twice, the iPSC colony edge cells begin to become round under a microscope and begin to separate from the surrounding spontaneous differentiated cells;

② digestion (differential digestion method): adding 2mL of 0.5mM EDTA digestive solution (AM 9260G, Saimer Feishell technology) into the cells treated in the step I, and incubating in an incubator; observing at intervals of 2min under a mirror until the iPSC colony is obviously separated from peripheral spontaneous differentiated cells, the cells in the iPSC colony become round and begin to fall off, and the form of the peripheral spontaneous differentiated cells is basically kept unchanged;

adding EDTA, incubating in an incubator, observing at intervals of 2min under a microscope, controlling digestion time in time to avoid more iPSC colony residues and spontaneous differentiated cell shedding, preferably, the digestion time is 3-5 min;

③ terminating digestion: at the moment, EDTA is abandoned, and E8 complete culture medium is added into the cells treated in the step two to stop digestion;

when the complete culture medium E8 is added to stop digestion, the culture medium is prevented from washing the culture surface;

collecting the iPSC-like removed spontaneous differentiated cells: gently flapping the bottom surface of the culture bottle after treatment, the iPSC colony falls off in a cell cluster shape, and the collected cell suspension can be used for iPSC passage; the spontaneously differentiated cells in the original flask were kept on the culture surface, and 10% FBS-containing high-glucose DMEM medium was added thereto, 5mL of each T25 flask was added, and the flask was placed in an incubator to continue the culture. Obtaining the spontaneous differentiated cells with iPSC removed;

preferably, the conditions for collecting the spontaneously differentiated cells from which the induced pluripotent stem cells are removed are 500-1000 rpm and centrifugation for 5 min; more preferably, the conditions for collecting the spontaneously differentiated cells from which the induced pluripotent stem cells are removed are 800 rpm and centrifugation is performed for 5 min.

(2) Results of the experiment

The results show that upon DPBS washing, iPSC colony edge cells begin to round and begin to separate from surrounding spontaneously differentiated cells (see figure 2); the degree of digestion of the EDTA digestive juice is shown in FIG. 3, and it can be seen that the iPSC colony is obviously separated from the surrounding spontaneous differentiated cells, the cells in the iPSC colony become round and begin to fall off, and the surrounding spontaneous differentiated cells basically keep unchanged in shape; the resulting iPSC depleted spontaneously differentiated cells were collected as shown in figure 4;

and after the iPSC is removed, the culture bottle is continuously placed in a culture box for culture, after 4-5 days, the spontaneous differentiated cells are obviously proliferated, but partial iPSC colonies can still be seen in the culture, when the iPSC colonies and the surrounding spontaneous differentiated cells have obvious limits, the operation of removing the iPS is carried out again, the method is the same as the above, and the operation can be carried out for 2-3 times to completely remove the iPSC-like cells.

5. Removal of epithelioid cells from spontaneously differentiated cells

(1) Experimental methods

When the cells in the culture bottle after the treatment do not have the iPSC-like colonies, the spontaneously differentiated cells can be seen to grow over the culture surface, and sometimes, part of the cells have epithelial-like cells, and differential digestion is carried out to passage by using different sensitivity degrees of the epithelial-like cells and fibroblasts to digestive juice so as to remove the epithelial-like cells, wherein the specific experimental operation is as follows:

washing: washing the obtained spontaneous differentiated cell DPBS without the iPSC twice, wherein each time lasts for 1 min; when the DPBS is washed twice, the DPBS must be washed sufficiently, and the culture medium residue on the vertical wall of the culture bottle is noticed;

secondly, digestion: adding 2mL of an accutase digestive solution diluted by 4 times into the washed cells; incubating at 37 ℃ for 6min, observing under a mirror, and observing under the mirror every 2min until the fibroblast-like cells become round and begin to fall off, and the shape of the epithelial-like cells is basically kept unchanged;

③ terminating digestion: at the moment, the accutase digestive juice is abandoned, and a high-sugar DMEM culture medium containing 10% FBS is added into the cells after digestion treatment to terminate digestion;

collecting spontaneously differentiated cells from which the epithelioid cells are removed: gently beating the bottom surface of the culture flask after digestion termination, collecting cell suspension, centrifuging at 800 rpm for 5min, discarding the supernatant, resuspending the cells in a high-glucose DMEM medium containing 10% FBS, adding 5mL into each T25 flask, and placing the bottles in an incubator for continuous culture to obtain the spontaneously differentiated cells without epithelioid cells.

(2) Results of the experiment

The results show that the fibroblast-like cells after 6min of digestion with accutase digest become rounded and begin to shed, while the epithelial-like cell morphology remains essentially unchanged (see fig. 5);

the epithelioid cells are not always present in the spontaneously differentiated cells, and if the spontaneously differentiated cells are free of the epithelioid cells, the subsequent cell expansion operation can be directly performed without this operation.

6. Expansion of spontaneously differentiated fibroblast-like cells

(1) Experimental methods

Washing: washing the obtained spontaneous differentiated cells without epithelioid cells twice with DPBS for 1min each time;

secondly, digestion: adding 2mL of an accutase digestive solution diluted by 4 times into the washed cells; incubating at 37 ℃ for 6min, observing under a mirror until more than 90% of the fibroblast cells become round and begin to fall off;

collecting the purified fibroblast-like cells: gently beating the bottom surface of the culture flask after digestion termination, collecting cell suspension, centrifuging for 5min at 800 rpm/min, discarding supernatant, resuspending cells with high-glucose DMEM medium containing 10% FBS, and carrying out passage 1: 3 to realize amplification of spontaneously differentiated fibroblast-like cells to obtain purified fibroblast-like cells.

(2) Results of the experiment

The results are shown in FIG. 6, which shows that the purified fibroblast-like cells are fibroblast-like in morphology and long spindle-shaped.

Example 2 immunofluorescence detection of fibroblasts derived from iPSC

1. Reverse transcription PCR

Extracting the purified total RNA of the fibroblast-like cells obtained in the example 1 by a Trizol method, and taking 1 mu g of the total RNA for reverse transcription PCR detection;

(1) collecting 200W cells, adding 1mL TRIZOL, extracting RNA, determining RNA concentration, inverting 1 μ g RNA to cDNA, and reverse transcription with kit

II Reverse transcription (all-type gold, AH101-02) according to manufacturer's instruction;

(2) use of

PCR Supermix (gold full-scale, AS111-11) was performed, premixed according to the reaction system described in Table 2;

TABLE 2 reaction System

(3) Then putting the system into a PCR instrument to react according to a 3-step method, wherein the cycle number is 45, and the reaction procedure is shown in Table 3;

TABLE 3 reaction procedure

The detection genes are vimentin (vimentin) and prolyl-4-hydroxylase beta (P4 beta) highly expressed by fibroblast; wherein, the sequence of the specific primer is as follows:

hOCT3/4 primer:

Forward Primer：5’-GACAGGGGGAGGGGAGGAGCTAGG-3’(SEQ ID NO:1)

Reverse Primer：5’-CTTCCCTCCAACCAGTTGCCCCAAAC-3’(SEQ ID NO:2)

the primer NONAG:

Forward Primer：5’-AAAGAATCTTCACCTATGCC-3’(SEQ ID NO:3)

Reverse Primer：5’-GAAGGAAGAGGAGAGACAGT-3’(SEQ ID NO:4)

VIMENTIN primers:

Forward Primer：5’-GACGCCATCAACACCGAGTT-3’(SEQ ID NO:5)

Reverse Primer：5’-CTTTGTCGTTGGTTAGCTGGT-3’(SEQ ID NO:6)

p4 β primer:

Forward Primer：5’-GGTGCTGCGGAAAAGCAAC-3’(SEQ ID NO:7)

Reverse Primer：5’-ACCTGATCTCGGAACCTTCTG-3’(SEQ ID NO:8)

GAPDH primer:

Forward Primer：5’-GGACTGAGGCTCCCACCTTT-3’(SEQ ID NO:9)

Reverse Primer：5’-GCATGGACTGTGGTCTGCAA-3’(SEQ ID NO:10)

2. immunofluorescence detection

Washing the purified fibroblast-like cells DPBS for three times, 3min each time; fixing by 4% PFA for 20min at room temperature; washing with DPBS for three times, each for 3 min; carrying out membrane permeation on 0.1% Triton-X100 for 10min at room temperature; washing with DPBS for three times, each for 3 min; sealing with 5% sheep serum at room temperature for 30 min; removing blocking solution by suction, adding primary antibody (1:100 dilution, rabbit anti-human VIMENTIN antibody) working solution; incubating overnight at 4 ℃; washing with DPBS for three times, each for 3 min; adding secondary antibody working solution (diluted 1:100, donkey anti-rabbit-IgG-Alexa)

488 antibody), incubated at room temperature for 1hr in the dark; washing with DPBS for three times, each for 3 min; staining the nucleus for 5min by Hoechst 33342; washing with DPBS for 3min three times, observing under fluorescent microscope andand (6) photographing.

3. Results of the experiment

The results of PCR detection show that iPSC spontaneously differentiates into fiber-like cells, which do not express specific transcription factors oct-4 and nanog of iPSC, and express vimentin (vimentin) and prolyl-4-hydroxylase beta (P4 beta) expressed by fibroblasts (see FIG. 7); results of immunofluorescence assays showed that vimentin was predominantly expressed in the cytoplasm of ipscs spontaneously differentiating into fiber-like cells (see fig. 8A-C).

Example 3 flow cytometry detection of fibroblasts derived from ipscs

1. Experimental methods

(1) Cell digestion

Removing the purified fibroblast-like cell culture solution obtained in example 1 by using a pipettor, adding calcium-magnesium-free PBS, slightly shaking the culture flask, discarding the calcium-magnesium-free PBS, and repeating the above process once; then adding Accutase diluted by 4 times, standing at 37 ℃, and observing the cell digestion condition under a microscope; when most of the cells are separated from the wall, the culture dish is tapped, and the cell suspension is collected into a 50mL centrifuge tube;

(2) single cell suspension preparation

Filtering the cell suspension in the 50mL centrifuge tube into a new 50mL centrifuge tube by using a 30-micron-aperture screen, collecting the cells at 300g multiplied by 5min, and discarding the supernatant;

(3) cell fixation

The collected cells are re-suspended and mixed evenly by 10mL of PBS without calcium and magnesium, 50 mu L of the mixture is sampled for counting the cells, the cells are washed once in 300g multiplied by 5min, and the supernatant is discarded; then re-suspending and mixing the mixture by using 10mL of calcium-magnesium-free PBS, washing the mixture once at 300g multiplied by 5min, and removing supernatant; adding 1mL of 4% paraformaldehyde solution, and fixing at room temperature for 30 min;

(4) sealing of

Centrifuging the fixed cells for 300g multiplied by 5min, discarding the supernatant, resuspending and mixing the cells uniformly by 1mL of calcium-magnesium-free PBS, washing the cells once for 300g multiplied by 5min, and discarding the supernatant; then re-suspending and mixing the mixture evenly with 1mL of PBS without calcium and magnesium, washing the mixture once at 300g multiplied by 5min, and discarding the supernatant; resuspending and mixing the mixture evenly with 1mL of calcium-magnesium-free PBS for the third time, washing the mixture once at 300g multiplied by 5min, and discarding the supernatant; adding 1mL of 1% BSA blocking solution, and blocking at room temperature for 30 min;

(5) fluorescent staining

Centrifuging the closed cells for 300g × 5min, discarding the supernatant, adding 0.7mL of calcium-magnesium-free PBS (phosphate buffer solution) to resuspend the cells, gently blowing and uniformly mixing, subpackaging the cells into 7 EP tubes with the volume of 1.5mL, keeping 1 EP tube as a control, adding the antibody into the rest 6 cells according to the proportion, incubating the cells in a refrigerator at 4 ℃ in a dark place for 30min, centrifuging for 300g × 5min, and discarding the supernatant; resuspending and mixing with 1mL of calcium-magnesium-free PBS, washing once at 300g multiplied by 5min, and discarding the supernatant; then re-suspending and mixing the mixture by using 1mL of calcium-magnesium-free PBS, washing the mixture once at 300g multiplied by 5min, and removing supernatant; resuspending and mixing the mixture evenly with 1mL of calcium-magnesium-free PBS for the third time, washing the mixture once at 300g multiplied by 5min, and discarding the supernatant; adding 200 mu L of calcium-magnesium-free PBS, gently blowing, beating and uniformly mixing, transferring to a flow cytometer tube for flow cytometer detection.

2. Results of the experiment

The results are shown in fig. 9A-C, and show that the purified fibroblast-like cells prepared in the invention do not express CD34 (positive rate of 0.218%), highly express CD105 (positive rate of 92.9%), partially express CD73 (positive rate of 17.9%), further indicating that the fibroblast cells prepared and separated successfully in the invention.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> Shino Rev medicine science and technology (New zone of Zhuhai horizontal organ) Co., Ltd

<120> a method for isolating fibroblasts derived from human induced pluripotent stem cells and uses thereof

<141> 2021-08-12

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Claims

1. A method for isolating fibroblasts from induced pluripotent stem cells, comprising the steps of:

(3) purifying the fibroblasts in the spontaneous differentiated cells obtained in the step (2) from which the induced pluripotent stem cells are removed to obtain purified fibroblasts;

the preparation of the induced pluripotent stem cells having spontaneously differentiated cells described in step (1) comprises the steps of:

the digestive juice in the step (a) is accutase digestive juice;

the culture medium in the step (a) is DMEM/F12 culture medium;

the culture medium in the step (b) is E8 culture medium;

the ROCK inhibitor in step (b) is Y27632;

the culture medium in the step (c) is E6 culture medium;

the ROCK inhibitor of step (c) is Y27632;

the induced pluripotent stem cells with spontaneously differentiated cells described in step (1) are derived from a human;

the removing process in the step (2) comprises the following steps: adding digestive juice into induced pluripotent stem cells with spontaneously differentiated cells, digesting and incubating until spontaneously differentiated cells exist around induced pluripotent stem cell colonies in a digestion product and obvious boundaries exist between the spontaneously differentiated cells and the induced pluripotent stem cell colonies, terminating digestion, removing the cells which can drop out from the digestion product, and collecting the spontaneously differentiated cells from which the induced pluripotent stem cells are removed;

the digestive juice is EDTA digestive juice;

the termination of digestion comprises discarding the digestion solution and adding a culture medium to the cells after digestion treatment;

the culture medium is E8 complete culture medium;

the purification process in the step (3) comprises the following steps: adding a digestive juice into the spontaneous differentiated cells from which the induced pluripotent stem cells are removed, digesting and incubating until the fibroblast-like cells become round and begin to fall off, terminating digestion, and collecting the purified fibroblasts;

the digestive juice is accutase digestive juice;

the culture medium is a high-glucose DMEM culture medium containing 10% FBS.

2. The method of claim 1, wherein the digest solution of step (a) is a preheated accutase digest solution at 37 ℃.

3. The method of claim 1, wherein the amount of digestive juice used in step (a) is 1 mL.

4. The method according to claim 1, wherein the culturing in step (a) is carried out at 37 ℃ and 5% CO₂。

5. The method according to claim 1, wherein the culturing time in step (a) is 2-3 min.

6. The method according to claim 1, wherein the medium in step (a) is pre-warmed DMEM/F12 medium at 37 ℃.

7. The method according to claim 1, wherein the amount of DMEM/F12 medium used in step (a) is 5 mL.

8. The method according to claim 1, wherein the centrifugation in step (b) is performed at 200g for 5 min.

9. The method according to claim 1, wherein the medium in step (b) is E8 medium preheated at 37 ℃.

10. The method according to claim 1, wherein the E8 medium is used in an amount of 600- & gt 700 μ L in step (b).

11. The method according to claim 1, wherein the amount of Y27632 used in step (b) is 10 μ M.

12. The method according to claim 1, wherein the medium in step (c) is E6 medium preheated at 37 ℃.

13. The method according to claim 1, wherein the amount of E6 medium used in step (c) is 3 mL.

14. The method according to claim 1, wherein the amount of Y27632 in step (c) is 10 μ M.

15. The method of claim 1, wherein the cell density in step (c) is 1.2 x 10⁴/cm²。

16. The method according to claim 1, wherein the culturing in step (c) is carried out at 37 ℃ and 5% CO₂。

17. The method of claim 1, wherein said culturing in step (c) further comprises repeating said changing each day.

18. The method of claim 1, wherein the number of passages in step (c) is 2-3.

19. The method according to claim 1, wherein the amount of EDTA digest used in step (2) is 0.5mM, 2 mL.

20. The method according to claim 1, wherein the digestion in step (2) is performed for 3-5 min.

21. The method according to claim 1, wherein the conditions for collecting the spontaneously differentiated cells from which the induced pluripotent stem cells are removed in step (2) are 500-1000 rpm and centrifugation for 5 min.

22. The method according to claim 21, wherein the conditions for collecting the spontaneously differentiated cells from which the induced pluripotent stem cells are removed in step (2) are 800 rpm and centrifugation is performed for 5 min.

23. The method of claim 1, further comprising washing the cells to be digested with a wash buffer prior to said digesting in step (2).

24. The method of claim 23, wherein the wash buffer comprises DPBS buffer, PBS buffer, TBS buffer, PE buffer, D-Hanks balanced salt buffer, or Earle's balanced salt buffer.

25. The method of claim 24, wherein the wash buffer is a DPBS buffer.

26. The method of claim 23, wherein the washing is a DPBS buffer wash 2 times for 1min each.

27. The method according to claim 1, wherein the amount of the accutase digest used in step (3) is 2mL at 4-fold dilution.

28. The method according to claim 1, wherein the digestion in step (3) is carried out for a period of 4-7 min.

29. The method according to claim 1, wherein the incubation in step (3) is carried out at 37 ℃ for 6 min.

30. The method as claimed in claim 1, wherein the collecting conditions of the purified fibroblasts in step (3) are 500-.

31. The method according to claim 30, wherein the collecting of the purified fibroblasts in step (3) is performed under conditions of 800 rpm and 5min of centrifugation.

32. The method of claim 1, further comprising washing the cells to be digested with a wash buffer prior to said digesting in step (3).

33. The method of claim 32, wherein the wash buffer comprises DPBS buffer, PBS buffer, TBS buffer, PE buffer, D-Hanks balanced salt buffer, or Earle's balanced salt buffer.

34. The method of claim 33, wherein the wash buffer is a DPBS buffer.

35. The method of claim 32, wherein the washing is a DPBS buffer wash 2 times for 1min each.

36. The method of claim 1, wherein the induced pluripotent stem cells with spontaneously differentiated cells are induced pluripotent stem cell colonies surrounded by at least 5% spontaneously differentiated cells, and the spontaneously differentiated cells are in the form of long spindle.

37. The method of claim 1, further comprising removing epithelioid cells from the spontaneously differentiated cells;

the removing of epithelioid cells from spontaneously differentiated cells comprises: adding digestive juice into the spontaneous differentiated cells from which induced pluripotent stem cells are removed, digesting and incubating until fibroblast-like cells become round and begin to fall off, keeping the shape of the epithelial-like cells basically unchanged, stopping digestion, and collecting the spontaneous differentiated cells from which the epithelial-like cells are removed;

the digestive juice is accutase digestive juice;

the digestion termination comprises the steps of discarding digestion solution and adding culture medium into cells after digestion treatment;

the culture medium is a high-glucose DMEM culture medium containing 10% FBS.

38. The method of claim 37, wherein the accutase digest is used in an amount of 2mL at 4-fold dilution.

39. The method of claim 37, wherein the digestion is for a period of 4-7 min.

40. The method of claim 37, wherein the incubation is at 37 ℃ for 6 min.

41. The method as claimed in claim 37, wherein the collecting conditions for collecting the spontaneously differentiated cells from which the epithelioid cells are removed are 500-.

42. The method of claim 41, wherein the collecting conditions to obtain the epithelioid-depleted spontaneously differentiated cells are 800 rpm and centrifugation for 5 min.

43. The method of claim 37, further comprising washing the cells to be digested with a wash buffer prior to said digesting.

44. The method of claim 43, wherein the wash buffer comprises DPBS buffer, PBS buffer, TBS buffer, PE buffer, D-Hanks balanced salt buffer, or Earle's balanced salt buffer.

45. The method of claim 44, wherein the wash buffer is a DPBS buffer.

46. The method of claim 43, wherein the washing is a DPBS buffer wash 2 times for 1min each.