Embryoid body digestion solution and its application in isolating hematopoietic stem cells

Abstract

The present invention relates to an embryoid body digestion solution and its application in isolating hematopoietic stem cells. Specifically, the present invention provides a digestion solution suitable for embryoid body dissociation, comprising two components, collagenase and dispase, which has a synergistic effect on the dissociation or digestion of embryoid bodies, and can efficiently dissociate embryoid bodies into single cells in a short time, and the obtained single cells can maintain a high cell viability, good cell phenotype and diversity of cell groups, which can be used for subsequent research or sorting target cells, such as obtaining hematopoietic stem cells. The present invention also provides a method and application for digesting embryoid bodies, which has broad application prospects.

Description

Technical Field

[0001] The invention relates to the field of cell culture, in particular to an embryoid body digestion solution and application thereof in separating hematopoietic stem cells. Background Art

[0002] Hematopoietic stem cells, as a type of adult stem cell, are located at the top of the hematopoietic system cascade and are considered to be the "ancestors" of various immune cells in the body. They have strong self-renewal ability and multidirectional differentiation potential. The hematopoietic stem cells in the human body are originally derived from the developing embryo, and have continued throughout a person's life. Hematopoietic stem cells continuously provide blood cells for the body. The earliest clinical application of hematopoietic stem cells is hematopoietic stem cell transplantation, which is used to treat blood system diseases such as leukemia and aplastic anemia. In recent years, with the continuous development of biomedical technology and the deepening of research on hematopoietic stem cells, hematopoietic stem cells and their derivatives have made significant progress and breakthroughs in organ transplantation, treatment of genetic diseases, treatment of immune system diseases, and tumor treatment. However, the insufficient number of hematopoietic stem cells and the need for HLA matching have always been the difficulties in their clinical application.

[0003] In 2006, Shinya Yamanaka's team at Kyoto University in Japan successfully induced induced pluripotent stem cells (iPSCs) in vitro and confirmed that they can differentiate into various types of cells in vitro, which opened a new chapter in basic research, drug development and personalized treatment. The advantages of iPSCs' unlimited in vitro expansion, pluripotency and ease of gene editing make them a source of various cell banks with unlimited potential. Therefore, obtaining hematopoietic stem cells by inducing differentiation of iPSCs in vitro has the hope of establishing a universal and sufficient hematopoietic stem cell bank, further expanding the scope of application of hematopoietic stem cells and their derivatives.

[0004] There are many ways to induce iPSC to differentiate into hematopoietic stem cells in vitro. In terms of differentiation form, it was initially based on 2D planar differentiation. By adding various related cytokines, iPSC cultured in a planar manner were induced to differentiate into hematopoietic stem cells. However, the yield of 2D differentiation is limited, and industrial production is difficult. As the research progressed, it was found that iPSCs, when cultured in suspension under certain conditions, can spontaneously form polymers called embryoid bodies (EBs). Embryoid bodies have a germ layer structure similar to that of an embryo, and can be directed to differentiate into characteristic cells under the action of exogenous cytokines. This differentiation based on embryoid bodies is considered to be closer to in vivo differentiation, and the 3D differentiation system is more conducive to the requirements of industrial production.

[0005] To obtain the required hematopoietic stem cells from embryoid bodies at a specific differentiation stage, currently, most commercially available general digestive enzymes are used. The development of these digestive enzymes is based on the application of adherent cell passage. Therefore, in the digestion of embryoid bodies at the tissue level, there are problems such as long digestion time and low digestion efficiency. Therefore, how to better obtain single cells with the highest efficiency from the embryoid body differentiation system suitable for industrial production, and further analyze and enrich hematopoietic stem cells, whether for the research and development and clinical application of hematopoietic stem cells themselves or their derivative cells, is extremely urgent.

[0006] Therefore, there is an urgent need in the art to develop a method for efficiently and quickly isolating single cells from embryoid bodies. Summary of the invention

[0007] The purpose of the present invention is to provide a method for efficiently and quickly separating single cells from embryoid bodies.

[0008] In a first aspect of the present invention, an embryoid body digestion solution is provided, wherein the embryoid body digestion solution is composed of the following components:

[0009] (i) collagenase at a concentration of 0.2-3.0 mg / mL;

[0010] (ii) dispase at a concentration of 0.15-2.5 mg / mL;

[0011] (iii) a solvent, wherein the solvent is a culture medium or a buffer.

[0012] In another preferred embodiment, the collagenase is type I collagenase.

[0013] In another preferred embodiment, the type I collagenase is derived from Clostridium histolyticum.

[0014] In another preferred embodiment, the dispase is dispase II.

[0015] In another preferred embodiment, the dispase II is derived from Bacillus polymyxa.

[0016] In another preferred embodiment, the concentration of the collagenase is preferably 0.4-2.0 mg / mL, more preferably 0.4-1.6 mg / mL, and most preferably 0.8-1.6 mg / mL.

[0017] In another preferred embodiment, the concentration of the dispase is preferably 0.25-2.0 mg / mL, more preferably 0.3-1.5 mg / mL, and most preferably 0.5-1 mg / mL.

[0018] In another preferred embodiment, the collagenase is type I collagenase, and its concentration is 0.8-1.6 mg / mL; and the dispase is dispase II, and its concentration is 0.5-1 mg / mL.

[0019] In another preferred embodiment, the collagenase is type I collagenase, and its concentration is 0.8 mg / mL; and the dispase is dispase II, and its concentration is 0.5 mg / mL.

[0020] In another preferred embodiment, the collagenase is type I collagenase, and its concentration is 1.6 mg / mL; and the dispase is dispase II, and its concentration is 1 mg / mL.

[0021] In another preferred embodiment, the collagenase is type I collagenase, and its concentration is 0.4 mg / mL; and the dispase is dispase II, and its concentration is 0.25 mg / mL.

[0022] In another preferred embodiment, the culture medium is a universal cell culture medium, a stem cell-specific culture medium, or an embryoid body-specific culture medium.

[0023] In another preferred embodiment, the culture medium is selected from the group consisting of DMEM, Modified NutriStem ® hPSC XFMedium, NutriStem ® hPSC XF Medium, StemSpan TM AOF Medium or STEMdiff TM APEL TM 2 Medium.

[0024] In another preferred embodiment, the buffer is a phosphate buffer.

[0025] In another preferred embodiment, the buffer is DPBS.

[0026] In another preferred embodiment, the digestion solution is used to dissociate embryoid bodies into single cells.

[0027] In a second aspect of the present invention, a method for digesting embryoid bodies to obtain single cells is provided, comprising the steps of:

[0028] The embryoid bodies are digested using the digestion solution described in the first aspect of the present invention to obtain a digested cell suspension.

[0029] In another preferred embodiment, the embryoid bodies are formed by culturing pluripotent stem cells.

[0030] In another preferred embodiment, the pluripotent stem cells include: embryonic stem cells and induced pluripotent stem cells (iPSCs).

[0031] In another preferred embodiment, the embryoid bodies are embryoid bodies at various differentiation stages, including hematopoietic stem cell differentiation stage, hematopoietic lineage differentiation stage, lymphocyte differentiation stage and / or NK cell differentiation stage.

[0032] In another preferred embodiment, the digestion treatment time is 1-4 hours, preferably 1.5-3 hours, and more preferably 2-2.5 hours.

[0033] In another preferred embodiment, during the digestion process, the digestion system is mixed by pipetting every 20-30 minutes.

[0034] In another preferred embodiment, the temperature of the digestion treatment is 37±5°C.

[0035] In another preferred embodiment, the cell suspension after digestion has one or more characteristics selected from the following group:

[0036] (Y1) undissociated EBs account for ≤10%, preferably ≤5%, and more preferably ≤1% of the total EBs;

[0037] (Y2) The viability of single cells is ≥60%, preferably ≥65%, and more preferably ≥70%;

[0038] (Y3) Cell type enrichment of single cells.

[0039] In another preferred embodiment, the undissociated embryoid bodies refer to cell clusters with a diameter greater than 100 μm.

[0040] In another preferred embodiment, the method further comprises the step of: sorting the digested cell suspension to obtain single cells of the target type.

[0041] In another preferred embodiment, the target type of single cell is a hematopoietic stem cell.

[0042] In another preferred embodiment, the digested cell suspension is subjected to CD34 sorting and enrichment to obtain CD34+ hematopoietic stem cells.

[0043] In another preferred embodiment, the purity of the enriched CD34+ hematopoietic stem cells is ≥85%, preferably ≥90%, and more preferably ≥95%.

[0044] In another preferred embodiment, the hematopoietic stem cells have the potential to differentiate into multiple cells, including: NK cells, T cells, megakaryocytes, or a combination thereof.

[0045] In a third aspect of the present invention, there is provided a use of a combination of active ingredients for preparing a reagent for digesting or dissociating embryoid bodies;

[0046] The active ingredient combination consists of (i) a first active ingredient: collagenase, and (ii) a second active ingredient: dispase.

[0047] In another preferred embodiment, the collagenase is type I collagenase, and the type I collagenase is derived from Clostridium histolyticum.

[0048] In another preferred embodiment, the dispase is dispase II, and the dispase II is derived from Bacillus polymyxa.

[0049] In another preferred embodiment, the concentration of collagenase in the reagent is 0.2-3.0 mg / mL, preferably 0.4-2.0 mg / mL, more preferably 0.4-1.6 mg / mL, and most preferably 0.8-1.6 mg / mL.

[0050] In another preferred embodiment, the concentration of dispase in the reagent is 0.15-2.5 mg / mL, preferably 0.25-2.0 mg / mL, more preferably 0.3-1.5 mg / mL, and most preferably 0.5-1 mg / mL.

[0051] In a fourth aspect of the present invention, a kit is provided, comprising:

[0052] (C1) a first container, wherein the first container contains the embryoid body digestion solution according to the first aspect of the present invention; and

[0053] Instructions or labels, wherein the instructions or labels indicate that the kit is used to digest embryoid bodies to obtain single cells.

[0054] In another preferred embodiment, the kit further comprises:

[0055] (C2) A second container containing a cell sorting reagent for sorting the cell suspension after the embryoid bodies are digested to obtain single cells of the target type.

[0056] In another preferred embodiment, the target type of single cell is a hematopoietic stem cell.

[0057] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0058] The beneficial effects of the present invention include:

[0059] 1. The digestion solution of the present invention can quickly and efficiently dissociate embryoid bodies to obtain a single cell suspension with a high viability, thereby obtaining a digestion solution that can be used for subsequent differentiation or research.

[0060] 2. The two enzymes in the digestion solution of the present invention can play a synergistic role, so that the single cells obtained by digestion can maintain a high cell viability, a good cell phenotype and a diversity of cell groups.

[0061] 3. The single cells obtained by the method of the present invention can be further sorted to obtain target cells, such as hematopoietic stem cells.

[0062] 4. The hematopoietic stem cells dissociated and sorted by the method of the present invention have the potential to differentiate into a variety of immune cells, and can be differentiated into various types of target cells, with good differentiation efficiency and high differentiation yield, which is conducive to further scientific research and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The embryoid bodies before digestion; Group 1: added digestion solution 1; Group 2: added digestion solution 2 with half the amount of enzyme; Group 3: added digestion solution 3 with double the amount of enzyme; Group 4: added digestion solution 4 dissolved in DMEM; Group 5: added digestion solution 5 dissolved in DPBS; Group 6: added digestion solution 6 containing only dispase; Group 7: added digestion solution 7 containing only collagenase; Group 8: added commercially available Accutase digestion solution; Group 9: added commercially available Tryple digestion solution; Group 10: added commercially available GCDR digestion solution, and the upper and lower two wells of each group are duplicate wells.

[0064] Figure 2 The embryoid bodies were digested with the digestion solution of each group for 2 hours.

[0065] Figure 3 The cell morphology under the microscope after 2 hours of digestion in each group of digestion solutions.

[0066] Figure 4 After each group of digestive fluids were digested for 2 hours, the size and number of particles in the digestive fluids were detected using a microfluidic imaging particle analysis system.

[0067] Figure 5 The cell viability was detected by flow cytometry after 2 hours of digestion in each group of digestion solutions.

[0068] Figure 6 The cell phenotypes detected by flow cytometry after 2 hours of digestion in each group of digestion solutions.

[0069] Figure 7 This is a cell clustering diagram obtained by single-cell sequencing of the single-cell suspension obtained by digestion with digestion solution 1.

[0070] Figure 8 This is the flow cytometry phenotype of the single cell suspension obtained by digestion with digestion solution 1 before and after enrichment with CD34 magnetic beads.

[0071] Fig. 9This is the flow cytometry phenotype of enriched CD34+ cells differentiated into CD3-CD56+ NK cells.

[0072] Fig.10 This is a graph showing the yield of CD34+ cells differentiated into CD3-CD56+ NK cells after enrichment.

[0073] Fig.11 This is the flow cytometry phenotype of the enriched CD34+ cells differentiated into CD8+ T cells.

[0074] Fig.12 This is a graph showing the yield of CD34+ cells differentiated into CD8+ T cells after enrichment.

[0075] Fig.13 This is the flow cytometry phenotype of the enriched CD34+ cells differentiated into CD41+CD42b+ megakaryocytes. DETAILED DESCRIPTION

[0076] After extensive and in-depth research, the inventors have developed a digestion solution that can quickly and efficiently dissociate embryoid bodies to obtain high-viability single cells, wherein the two components of collagenase and dispase contained therein have a synergistic effect on the dissociation or digestion of embryoid bodies, and can efficiently dissociate embryoid bodies into single cells in a short time, and the obtained single cells can maintain a high cell viability, good cell phenotype and diversity of cell groups, which can be used for subsequent research or sorting target cells, such as obtaining hematopoietic stem cells. The digestion effect of the digestion solution of the present invention is better than that of commercially available digestion solutions and digestion solutions with a single enzyme component, and is suitable for the dissociation of embryoid bodies at various differentiation stages.

[0077] On this basis, the present invention has been completed.

[0078] Embryoid body

[0079] Embryoid bodies are spherical structures formed by stem cells under certain in vitro culture conditions. They have three germ layers: endoderm, mesodermal and ectoderm. They are morphologically very similar to the early embryonic development stage of mammals. Embryoid bodies have the ability to self-renew and have multidirectional differentiation potential. They can differentiate into almost all types of cells in the body, including nerve cells, heart cells, liver cells, kidney cells, etc. Therefore, embryoid bodies have important application value in regenerative medicine, tissue engineering and disease model research.

[0080] In the present invention, the source of the embryoid body is not limited, and may be derived from, for example, embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC).

[0081] In the present invention, the differentiation stage of the embryoid body is not limited, and the differentiation stage includes any intermediate stage in the process of embryoid body differentiation into various types of cells after iPSC forms embryoid body, such as hematopoietic stem cell differentiation stage, hematopoietic lineage differentiation stage, lymphocyte differentiation stage, NK cell differentiation stage, etc.

[0082] Embryoid body digestion solution of the present invention

[0083] There are rich types of cell populations in embryoid bodies. In order to separate specific cell populations from embryoid bodies, the embryoid bodies need to be digested or dissociated first to obtain single cells.

[0084] As used herein, the terms "digestion" and "dissociation" are used interchangeably and both refer to the process of converting embryoid bodies from a cell mass state to a single cell state.

[0085] In the present invention, a digestion solution suitable for digesting embryoid bodies into single-cell state is provided, and the components of the digestion solution include collagenase, dispase and a solvent.

[0086] As used herein, collagenase, also known as collagenase, can specifically hydrolyze the three-dimensional helical structure of natural collagen under physiological pH and temperature conditions without damaging other proteins and tissues. In a preferred embodiment, the collagenase is derived from Clostridium histolyticum ( Clostridium histolyticum In a preferred embodiment, the collagenase used in the digestive juice of the present invention is type I collagenase.

[0087] As used herein, dispase is a non-specific metalloproteinase, a fast, effective and mild cell digestion enzyme that causes little damage to cells and can maintain cell membrane integrity; it has strong stability and is not affected by temperature, pH and serum components.

[0088] In the present invention, an embryoid body digestion solution containing both collagenase and dispase is provided for the first time, wherein the concentration of the collagenase is 0.2-3.0 mg / mL, preferably 0.4-2.0 mg / mL, preferably 0.6-2.0 mg / mL, more preferably 0.4-1.6 mg / mL, and most preferably 0.8-1.6 mg / mL; the concentration of the dispase is 0.15-2.5 mg / mL, preferably 0.25-2.0 mg / mL, more preferably 0.3-1.5 mg / mL, and most preferably 0.5-1 mg / mL.

[0089] In the digestion solution of the present invention, the type of solvent can be culture medium or buffer, including any culture medium or buffer suitable for embryoid body cell culture.

[0090] In a preferred embodiment, the culture medium includes: a general cell culture medium, a stem cell-specific culture medium, or an embryoid body-specific culture medium, which can be adjusted according to different stages of embryoid body differentiation.

[0091] For example, the universal culture medium includes DMEM culture medium; the dedicated culture medium includes: NutriStem ® hPSCXF Medium (Sartorius, Cat: 05-100-1A) or ModifiedNutriStem ® hPSC XF Medium (Sartorius, CAT: 06-5100-01-1A) or StemSpan TM AOF Medium (Stemcell, CAT: 100-0130) or STEMdiff TM APEL TM 2 Medium (Stemcell, CAT: 05275), but not limited to.

[0092] The embryoid bodies to be digested in the present invention may be at any stage of differentiation. Different differentiation stages require different culture media and cytokines according to requirements. Accordingly, the solvent used to prepare the embryoid body digestion solution needs to adopt the corresponding culture system during differentiation.

[0093] In a preferred embodiment, the buffer may be a phosphate buffer, such as DPBS, but is not limited thereto.

[0094] In the digestive juice of the present invention, the type of solvent does not affect the effects of the collagenase and dispase.

[0095] Method for obtaining single cells from embryoid bodies

[0096] In the present invention, a method for obtaining a single cell from an embryoid body is provided, comprising the steps of: digesting the embryoid body with the digestion solution of the present invention.

[0097] The digestion solution of the present invention can be used to digest the embryoid bodies into a single cell state in a very short time. In a preferred embodiment, the digestion treatment time is 1-4 hours, preferably 1.5-3 hours, more preferably 2-2.5 hours, for example 2 hours. Preferably, during the digestion treatment process, the digestion system is blown and mixed every 20-30 minutes to accelerate the digestion process.

[0098] In a preferred embodiment, in the cell suspension obtained after treatment with the digestion solution of the present invention, undissociated embryoid bodies account for ≤10%, preferably ≤5%, and more preferably ≤1% of the total embryoid bodies; the undissociated embryoid bodies refer to cell clusters with a diameter greater than 100 μm.

[0099] In a preferred embodiment, in the cell suspension obtained after being treated with the digestion solution of the present invention, the viability of single cells is ≥60%, preferably ≥65%, and more preferably ≥70%.

[0100] In a preferred embodiment, in the cell suspension obtained after being treated with the digestion solution of the present invention, the single cell types are rich, and the proportion of each phenotype is close to the actual proportion in the embryoid body.

[0101] application

[0102] The present invention also provides the use of the embryoid body digestion solution of the present invention, which is used to separate the embryoid body into a single cell state, and further sort and enrich the cells to analyze the cells in the embryoid body, or obtain the target cell population in the embryoid body.

[0103] In some embodiments, the single-cell suspension obtained by digesting embryoid bodies with the digestion solution of the present invention can be used for scientific research purposes, such as performing analytical experiments such as single-cell sequencing.

[0104] In some embodiments, cells can be sorted and enriched from the single cell suspension obtained by digestion, and the sorting method can be any method known in the art, such as flow cytometer sorting.

[0105] In a preferred embodiment, the target cells are hematopoietic stem cells, which are enriched by CD34 magnetic beads to obtain CD34+ hematopoietic stem cells. The separated hematopoietic stem cells can be used for subsequent purposes, such as differentiating into different target cells such as NK cells, T cells, megakaryocytes, etc. under the action of corresponding cytokines.

[0106] In a preferred embodiment, the hematopoietic stem cells dissociated and sorted using the method of the present invention have been proven to be able to differentiate into various types of target cells with good differentiation efficiency and high differentiation yield. A large number of CD34+ hematopoietic stem cells can be digested and sorted in batches to obtain a hematopoietic stem cell bank, which can serve as a reserve of the source of hematopoietic stem cells and their derivative cells.

[0107] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are weight percentages and weight fractions.

[0108] Example 1: Preparation of different digestive juices and comparison of digestive effects

[0109] In this example, 7 different digestion solutions were prepared for tissue dissociation of embryoid bodies during the differentiation of iPSCs into blood lineage cells. The embryoid body digestion solution comprises type I collagenase, dispase II and embryoid body differentiation medium, wherein the embryoid body differentiation medium is adjusted according to different differentiation stages.

[0110] 1. Preparation of digestive juice

[0111] The ratio and dosage of collagenase and dispase in each digestion solution and the type of culture medium are shown in Table 1 below.

[0112] Table 1 Components and dosage of each digestive fluid

[0113]

[0114] The digestion solution was prepared according to the components and dosages in Table 1, as follows:

[0115] Digestion Solution 1 (Complete Reagent):

[0116] Preparation of embryoid body digestion solution: Weigh 16 mg collagenase (Sigma, Cat: C0130-1G) and 10 mg dispase (Gibco, Cat: 17105041) in 20 mL ModifiedNutriStem ® After fully dissolving and mixing in hPSC XF Medium (Sartorius, CAT: 06-5100-01-1A), filter with a 0.22 μm filter membrane and place in a 4°C refrigerator for later use.

[0117] Digestive solution 2 (half the amount of enzyme):

[0118] Preparation of embryoid body digestion solution with half the amount of enzyme compared to digestion solution 1: weigh 8 mg collagenase (Sigma, Cat: C0130-1G) and 5 mg dispase (Gibco, Cat: 17105041) in 20 mL Modified NutriStem® After fully dissolving and mixing in hPSC XFMedium (Sartorius, CAT: 06-5100-01-1A), filter with a 0.22 μm filter membrane and place in a 4°C refrigerator for later use.

[0119] Digestion Solution 3 (double the amount of enzyme):

[0120] Preparation of embryoid body digestion solution with double the amount of enzyme compared to digestion solution 1: weigh 32 mg collagenase (Sigma, Cat: C0130-1G) and 20 mg dispase (Gibco, Cat: 17105041) in 20 mL Modified NutriStem ® After fully dissolving and mixing in hPSC XFMedium (Sartorius, CAT: 06-5100-01-1A), filter with a 0.22 μm filter membrane and place in a 4°C refrigerator for later use.

[0121] Digestion Solution 4 (Complete Reagent / DMEM):

[0122] Compared with digestion solution 1, the culture medium was replaced with DMEM: 16 mg collagenase (Sigma, CAT: C0130-1G) and 10 mg dispase (Gibco, CAT: 17105041) were weighed into 20 mL DMEM (Gibco, CAT: 11995065), fully dissolved and mixed, filtered with a 0.22 μm filter membrane, and placed in a 4°C refrigerator for later use.

[0123] Digestion Solution 5 (Complete Reagent / DPBS):

[0124] Compared with digestion solution 1, the culture medium was replaced with DPBS: 16 mg collagenase (Sigma, CAT: C0130-1G) and 10 mg dispase (Gibco, CAT: 17105041) were weighed into 20 mL DPBS (Gibco, CAT: 14200075), fully dissolved and mixed, filtered with a 0.22 μm filter membrane, and placed in a 4°C refrigerator for later use.

[0125] Digestion Solution 6 (Dispase only):

[0126] Preparation of embryoid body digestion solution containing only dispase compared to digestion solution 1: weigh 10 mg dispase (Gibco, CAT: 17105041) in 20 mL Modified NutriStem ®After fully dissolving and mixing in hPSC XF Medium (Sartorius, CAT: 06-5100-01-1A), filter with a 0.22 μm filter membrane and place in a 4°C refrigerator for later use.

[0127] Digestion Solution 7 (Collagenase only):

[0128] Preparation of embryoid body digestion solution containing only collagenase compared to digestion solution 1: weigh 16 mg collagenase (Sigma, Cat: C0130-1G) in 20 mL Modified NutriStem ® After fully dissolving and mixing in hPSC XF Medium (Sartorius, CAT: 06-5100-01-1A), filter with a 0.22 μm filter membrane and place in a 4°C refrigerator for later use.

[0129] In addition to the above 7 homemade digestion solutions, this example also provides 3 commercially available digestion solutions: Accutase Cell Detachment Solution (Innovative Cell Technologies, CAT: AT-104), TrypLE TM Select (ThermoFisher Scientific, CAT: 12563) and GCDR (Gentle Cell Dissociation Reagent, Stemcell, CAT: 100-0485) were used as controls. Accutase is a cell digestion solution containing proteolytic enzyme and collagenase activity, and TrypLE TM Select is a non-animal derived recombinant enzyme, GCDR is a chemically defined reagent that does not contain enzymes or proteins. The three commercially available digestion solutions are all commonly used digestion solutions in the field.

[0130] 2. Digestion Steps

[0131] The above-mentioned homemade digestion solution and commercially available digestion solution were used to digest the embryoid bodies at a specific differentiation period. The specific operation is as follows:

[0132] Embryoid bodies at the stage of hematopoietic stem cell differentiation (about differentiation day 9-15) were obtained and evenly plated in 6-well plates. They were divided into 10 groups, with 2 replicate wells in each group.

[0133] Each group was digested by adding different types of digestive fluids, e.g. Figure 1As shown, groups 1 to 7 correspond to homemade digestion solutions 1 to 7, respectively, and groups 8 to 10 correspond to commercially available digestion solutions Accutase, TrypLE, and GCDR, respectively. There are 20 wells in the above 10 groups, 2 mL of the corresponding digestion solution is added to each well, and the wells are placed in a 37°C incubator for digestion. Every 30 minutes, the wells are taken out and placed in a clean bench for mixing by pipetting. The digestion lasts for 2 hours.

[0134] 3. Digestion Results

[0135] After 2 h of digestion, take out the 6-well plate and take pictures ( Figure 2 ), while observing and recording the cell morphology under a microscope ( Figure 3 ).

[0136] The results showed that in the groups using digestion solution 1 (group 1), digestion solution 3 with double the amount of enzyme (group 3), digestion solution 4 dissolved in DMEM (group 4) and digestion solution 5 dissolved in DPBS (group 5), the embryoid bodies were completely digested into single-cell suspensions.

[0137] In the groups using digestion solution 2 (Group 2) with half the amount of enzyme and digestion solution 7 (Group 7) containing only collagenase, a small number of cell clumps were visible under the microscope; the tissue dissociation effect of digestion solution 6 (Group 6) containing only dispase was poor, and there were still many incompletely dissociated embryoid bodies.

[0138] The tissue dissociation effects of the groups using three commercially available digestive solutions, Accutase, TrypLE, and GCDR (Groups 8, 9, and 10), were poor. There were many undissociated embryoid bodies both under the naked eye and under the microscope, and the number of released single cells was relatively small.

[0139] The above results show that under the condition of a dissociation time of 2 hours, the digestion solutions using a combination of 0.8 mg / mL collagenase + 0.5 mg / mL dispase and a combination of 1.6 mg / mL collagenase + 1 mg / mL dispase have the best dissociation effect, and the dissociation effect is not affected by the solvent, and is better than the digestion solution containing only collagenase or dispase and the commercially available digestion solution.

[0140] Example 2: Detection of the number of single cells and undigested cell aggregates in the digested suspension

[0141] The size and number of single cells and undigested cell agglomerates in the 10 groups of suspensions obtained by digestion in Example 1 were detected using a microfluidic imaging particle analysis system (MFI), and the number of completely digested single cells with a diameter less than 25 μm was counted, and the number of cell agglomerates with a diameter greater than 100 μm was counted. The four groups (groups 6, 8, 9, and 10) with poor digestion effects were not tested because the undissociated embryoid bodies were larger and more numerous to avoid clogging the instrument pipeline.

[0142] The results are as follows Figure 4 As shown, there is no significant difference in the number of single cells obtained by digestion in the 6 groups tested; as for cell clumps, the number of cell clumps with a diameter greater than 100 μm obtained by digestion of embryoid body digestion solution, embryoid body digestion solution with doubled enzyme amount, embryoid body digestion solution dissolved in DMEM, and embryoid body digestion solution dissolved in DPBS is relatively small (about 1000), and the cell clumps in the suspension obtained by digestion of embryoid body digestion solution with half enzyme amount are relatively more, but there is no significant difference, and the cell clumps in the suspension obtained by digestion of embryoid body digestion solution containing only collagenase are significantly increased (about 4000), and there is a significant difference, which is about 2 to 4 times that of other groups. It shows that the digestion effect of digestion solution 1 to 5 containing both collagenase and dispase of the present invention is better than that of other groups.

[0143] Example 3: Viability detection of single cells obtained by digesting embryoid bodies with each group of digestion solutions

[0144] The viability of single cells obtained by digesting embryoid bodies with each group of digestion solutions in Example 1 was detected, and cell activity was detected by flow cytometry using Annexin V / 7-AAD staining, wherein Annexin V-7-AAD- characterized living cells.

[0145] The results are as follows Figure 5 As shown, the single cell viabilities obtained by digestion with digestion solution 1 and digestion solution 2 with half the amount of enzyme were the highest, the cell viabilities obtained by digestion with digestion solution 3 with double the amount of enzyme, digestion solution 4 dissolved in DMEM, and digestion solution 5 dissolved in DPBS all decreased, the cell viabilities obtained by digestion with digestion solution 6 containing only collagenase and digestion solution 7 containing only dispase all decreased significantly, and similarly, the single cell viabilities obtained by digestion with the three commercially available digestion solutions decreased significantly.

[0146] Example 4: Cell phenotype detection of single cells obtained by digesting embryoid bodies with each group of digestion solutions

[0147] The cell phenotype of the single cells obtained from the embryoid bodies digested by the digestion solution of each group in Example 1 was detected: the single cells obtained from the digestion of each group were stained with CD31, CD34, CD43, CD45, and CD144 antibodies, and flow cytometry was performed.

[0148] The results are as follows Figure 6As shown, the proportions of cell populations positive for each antibody in the groups with relatively complete digestion (groups 1, 2, 3, 4, 5, and 7) are relatively similar, while in the groups with incomplete digestion (groups 6, 8, 9, and 10), the proportions of various types of cells are more deviated from the former, and these deviations are most likely due to the different digestion times required for various types of cells. Incomplete digestion leads to a decrease in the proportion of some difficult-to-digest cell populations, such as cells in cell clumps larger than 100 μm that are not digested, so the proportion of easily digestible cell populations increases. The above results show that poor embryoid body digestion will not only lead to a significant decrease in the viability of single cells obtained by digestion, but also lead to deviations in cell phenotype identification and determination of different cell proportions.

[0149] Example 5: Single-cell transcriptome sequencing of the digested single-cell suspension

[0150] The single cell suspension obtained by digestion with digestion solution 1 in Example 1 was subjected to single cell transcriptome sequencing. The cell sample obtained by digestion with digestion solution 1 can fully meet the sample quality detection standard required for single cell transcriptome sequencing.

[0151] like Figure 7 As shown, after dimensionality reduction analysis of the sequencing results, it can be seen that the single cells digested from the embryoid bodies contain a variety of different cell types, and even cell populations with a relatively small proportion (such as Cluster 13, etc.) are also identified.

[0152] This shows that using digestion solution 1 to digest embryoid bodies can not only maintain good cell viability and cell phenotype, but also well maintain the diversity of cell populations, especially protecting rare cell populations, which is of great significance for subsequent in-depth analysis and research.

[0153] Example 6: In vitro differentiation ability of hematopoietic stem cells obtained by digestion

[0154] The single cell suspension digested with digestion solution 1 for 2 h in Example 1 was sorted, and forward sorting was performed using CD34+ magnetic beads (Miltenyi, CAT: 130-046-702), and the sorted cells were subsequently differentiated into NK cells, T cells and megakaryocytes.

[0155] The results of sorting and enrichment are as follows Figure 8 As shown in the figure, after magnetic bead sorting, CD34+ cells were significantly enriched, with a positive rate of more than 95%, and hematopoietic stem cells with considerable purity were obtained.

[0156] The results of the sorted cells differentiating into NK cells are as follows Fig. 9 and 10As shown, the yield of subsequent NK cell differentiation using this CD34+ hematopoietic stem cell can reach more than 100 times, and the differentiation positive rate (CD3-CD56+%) can reach more than 95%.

[0157] The results of the sorted cells differentiating into T cells are as follows Fig.11 and 12 As shown, the CD34+ hematopoietic stem cells obtained by magnetic bead sorting can be differentiated into T cells, and their yield can reach more than 200 times, and the differentiation positive rate (CD8+%) can reach more than 90%.

[0158] The results of the sorted cells differentiating into megakaryocytes are as follows Fig.13 As shown, the CD34+ hematopoietic stem cells obtained by magnetic bead sorting were subsequently differentiated into megakaryocytes, and megakaryocytes (CD41+CD42b+) were successfully obtained.

[0159] discuss

[0160] According to the results of the examples, the embryoid body digestion solution containing collagenase and dispase of the present invention can quickly and effectively digest the embryoid bodies, and the single cells obtained by digestion can maintain a high cell viability, a good cell phenotype and the diversity of cell populations. In comparison, both halving the amount of enzyme and doubling the amount of enzyme can maintain the digestion effect to a certain extent, so the concentration of the enzyme can be adjusted within a certain range.

[0161] However, the digestion effect of the digestion solution with only collagenase or only dispase added was obviously inferior to that of the complete embryoid body digestion solution, indicating that the two proteases can play a synergistic role to a large extent.

[0162] The three commercially available digestion solutions have poor effects on digesting embryoid bodies, which are manifested by incomplete digestion, low cell viability, deviation in cell phenotype identification, etc., and are not suitable for digesting embryoid bodies.

[0163] After dissociating the embryoid bodies using the embryoid digestion solution containing collagenase and dispase of the present invention, CD34+ hematopoietic stem cells with sufficient purity can be sorted, which provides the possibility for the industrial production of hematopoietic stem cells derived from iPSC. The hematopoietic stem cells are used for subsequent differentiation, and it has been confirmed that various types of target cells can be differentiated, and the differentiation efficiency is good and the differentiation yield is high, which is conducive to further scientific research and clinical application. By using this method to digest the embryoid bodies, a large number of CD34+ hematopoietic stem cells can be digested and sorted in batches to establish a hematopoietic stem cell bank, which can be used as a reserve of the source of hematopoietic stem cells and their derivative cells.

[0164] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for obtaining single cells by digesting embryoid bodies, characterized in that: Includes steps: Using embryoid body digestion solution to digest embryoid bodies, wherein the embryoid bodies are embryoid bodies derived from induced pluripotent stem cells (iPSCs), and the embryoid bodies are embryoid bodies in the hematopoietic stem cell differentiation stage; Wherein, the embryoid body digestion solution is composed of the following components: (i) collagenase, wherein the collagenase is type I collagenase and has a concentration of 0.4-0.8 mg / mL; (ii) dispase, wherein the dispase is dispase II and has a concentration of 0.25-0.5 mg / mL; (iii) a solvent, wherein the solvent is a culture medium, and the culture medium is ModifiedNutriStem ® hPSC XF Medium; The digestion treatment time is 1-4 hours to obtain a digested cell suspension, wherein the undissociated embryoid bodies account for ≤5% of the total amount of embryoid bodies, and the undissociated embryoid bodies refer to cell clusters with a diameter greater than 100 μm; The cell suspension contains single cells, and the digested cell suspension is sorted to obtain single cells of the target type; the sorting is to perform CD34 sorting and enrichment on the digested cell suspension to obtain CD34+ hematopoietic stem cells.

2. The method according to claim 1, characterized in that In the embryoid body digestion solution, the concentration of the collagenase is 0.8 mg / mL; and the concentration of the dispase is 0.5 mg / mL.

3. The method according to claim 1, characterized in that In the embryoid body digestion solution, the concentration of the collagenase is 0.4 mg / mL; and the concentration of the dispase is 0.25 mg / mL.

4. The method according to claim 1, characterized in that The digestion treatment time is 1.5-3 hours.

5. The method according to claim 4, characterized in that The purity of CD34+ hematopoietic stem cells after enrichment is ≥90%.

6. The method according to claim 1, characterized in that In the cell suspension after digestion, the viability of single cells is ≥70%.

7. A kit, characterized in that: The kit comprises: (C1) A first container, wherein the first container contains embryoid body digestion solution, wherein the embryoid body digestion solution is composed of the following components: (i) collagenase, wherein the collagenase is type I collagenase and has a concentration of 0.4-0.8 mg / mL; (ii) dispase, wherein the dispase is dispase II and has a concentration of 0.25-0.5 mg / mL; (iii) a solvent, wherein the solvent is a culture medium, and the culture medium is ModifiedNutriStem ® hPSC XF Medium; as well as An instruction manual or label indicating that the kit is used to digest embryoid bodies to obtain single cells, wherein the embryoid bodies are embryoid bodies derived from induced pluripotent stem cells (iPSCs), and the embryoid bodies are embryoid bodies at the hematopoietic stem cell differentiation stage; The kit also includes: (C2) A second container, wherein the second container contains a cell sorting reagent, which is used to sort the cell suspension after digestion of the embryoid bodies to obtain single cells of the target type; the single cells of the target type are hematopoietic stem cells, and the sorting is to perform CD34 sorting and enrichment on the cell suspension after digestion to obtain CD34+ hematopoietic stem cells.

8. The kit according to claim 7, characterized in that The hematopoietic stem cells have the potential to differentiate into multiple cells, including: NK cells, T cells, megakaryocytes, or a combination thereof.

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