Cartilage tissue single cell extraction kit and extraction method thereof

Abstract

The present application relates to a kind of extraction kit of cartilage tissue single cell and its extraction method, the kit includes: (1) first enzymolysis enzyme, the first enzymolysis enzyme includes one or more of trypsin, chondroitinase ABC and fucosidase;(2) second enzymolysis enzyme, the second enzymolysis enzyme includes collagenase II, neutral protease II, hyaluronidase and Dnase I;(3) base liquid, the base liquid includes base medium and / or buffer solution.The present application also provides the method for extracting single cell from cartilage tissue using the kit.The cartilage tissue single cell suspension prepared by the present application has the advantages of high cell yield and high activity, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and single-cell omics, specifically to a kit for extracting single cells from cartilage tissue and a method thereof. Background Technology

[0002] High-throughput sequencing technology can sequence hundreds of thousands to millions of DNA molecules in parallel at once. It has the advantages of high throughput, fast detection speed, flexibility, and low cost. It can accurately analyze the composition information of sample cells. Combined with high-throughput sequencing, it can reveal the gene structure and gene expression status of individual cells on a large scale, and clearly reflect the heterogeneity between cells.

[0003] The implementation of high-throughput sequencing technology and the acquisition of reliable results depend on the extraction of high-quality single-cell products from biological tissues.

[0004] Depending on the research objective and the research subject, different tissues are used for extracting single cells. Examples of tissues used for single-cell extraction include brain tissue, tumor tissue, adipose tissue, skin tissue, and bone tissue. Different tissues present different extraction challenges, thus requiring different solutions. For example, the challenge with brain tissue lies in the fragility of neurons and the adhesion of glial cells, necessitating a focus on addressing glial cell adhesion while employing gentle extraction conditions. The challenge with tumor tissue is the encapsulation of tumor cells by stromal cells, requiring the use of multiple enzymes in combination. The challenge with adipose tissue is lipid droplet interference and the difficulty in dissociating fibroblasts, requiring centrifugation to remove the fat layer after enzymatic digestion. The challenge with skin tissue is the presence of the stratum corneum barrier, requiring lengthy pretreatment.

[0005] Extracting single cells from bone tissue is the most difficult part of these tissues, mainly for the following reasons.

[0006] Bone tissue possesses a hardened, mineralized extracellular matrix. A key characteristic of bone tissue is its highly mineralized (primarily composed of hydroxyapatite crystals) extracellular matrix, which forms a rigid physical barrier, tightly encapsulating cells, especially deeply embedded bone cells. This makes it difficult for enzymes or chemical reagents to effectively penetrate and digest the matrix to release cells. Consequently, bone tissue exhibits low cell release efficiency and yield; drastic physical or chemical treatments are required, but such treatments can easily damage cell viability and integrity.

[0007] Bone tissue exhibits heterogeneity and fragility across cell types. It contains various cell types (osteoblasts, osteocytes, osteoclasts, bone marrow mesenchymal stem cells, endothelial cells, immune cells, etc.), each with significantly different tolerances to the separation process. Osteocytes, deeply embedded in lacunae and tubular networks, are the most difficult to release, and their elongated processes are highly susceptible to breakage during separation. Osteoclasts, large, multinucleated, and highly adherent, are relatively fragile and prone to death or rupture during separation. Osteoblasts / precursor cells are relatively easy to release, but can still be affected by harsh treatment. Therefore, single-cell suspensions obtained from bone tissue often fail to accurately reflect the cellular composition of the original tissue; certain key cell types (especially osteocytes and osteoclasts) may be lost or underrepresented, and cell viability may be low.

[0008] Bone tissue presents particular challenges in maintaining cell viability. Extracting single cells from bone tissue requires overcoming the time required to work through the rigid matrix (e.g., prolonged decalcification) and the intensity of processing (violent shaking, grinding, strong acids / chelating agents), which significantly impairs cell membrane integrity and metabolic activity. Nucleases and intracellular substances released from dead cells contaminate the background, interfering with downstream analysis. The consequences include low cell viability, affecting the quality of single-cell sequencing libraries (high dead cell background, low gene detection rate), and the potential for altered cell functional status due to stress.

[0009] Bone tissue is difficult to effectively dissociate and avoid aggregation. While releasing cells from bone tissue, it is necessary to fully dissociate it into single-cell states, avoiding duplexes or multicellular aggregates. Bone tissue digestion easily produces debris, extracellular matrix residues, and cell aggregates. Duplexes can be mistaken for hybrid cells in single-cell analysis, leading to incorrect biological interpretation, while debris can clog microfluidic chips or affect cell sorting efficiency.

[0010] Another challenge in single-cell extraction from bone tissue is removing debris. Bone fragments, mineral particles, and dissociated extracellular matrix also generate a large amount of background impurities, resulting in a reduced proportion of target cells and debris interfering with cell sorting and analysis.

[0011] Among these bone tissues, single-cell extraction of cartilage tissue, such as articular cartilage, has significant application value in basic research, disease mechanism analysis, and regenerative medicine. However, single-cell extraction of cartilage tissue also faces the following technical challenges: (1) low cell density, with cartilage cells accounting for only 1% to 5% of the tissue volume and containing a large amount of collagen and proteoglycan matrix; (2) dense extracellular matrix (ECM) hinders enzyme penetration, and type II collagen and aggregated proteoglycan form a rigid network, limiting the diffusion of digestive enzymes; (3) fragile cells are easily damaged, and mechanical shearing or oxidative stress during enzymatic digestion can lead to cell death. Therefore, the above-mentioned problems faced by single-cell extraction of bone tissue are particularly prominent in single-cell extraction of cartilage tissue, posing significant challenges in balancing cell viability and yield, fragment interference, and single-cell sequencing compatibility.

[0012] Although there are currently general methods for extracting single cells from various tissues, including methods for extracting single cells from cartilage tissue, these methods cannot effectively solve the aforementioned technical difficulties, and the extraction results are unsatisfactory.

[0013] Therefore, there is an urgent need for more optimized or new methods to solve the above problems in order to provide higher quality single-cell products of cartilage tissue, providing high-quality cell samples for subsequent scientific research analysis, such as single-cell sequencing, flow cytometry, cell culture and functional studies, or other applications. Summary of the Invention

[0014] To address the deficiencies in the prior art, this application provides a single-cell extraction kit for cartilage tissue in a first aspect, the extraction kit comprising:

[0015] (1) The first enzyme for hydrolysis, wherein the first enzyme for hydrolysis includes one or more of trypsin, chondroitin ABC and fucosidase;

[0016] (2) The second enzyme for hydrolysis, which includes collagenase II, neutral protease II, hyaluronidase and Dnase I;

[0017] (3) Base solution, which includes base culture medium and / or buffer solution.

[0018] In a second aspect, the present invention provides a method for extracting single cells from cartilage tissue, wherein the extraction method uses the extraction kit described in the first aspect of the present invention.

[0019] Compared with existing technologies, the present invention has the following advantages when used for single-cell extraction of cartilage tissue:

[0020] (1) High quality of single-cell products: cell viability can reach up to 93.34%, nucleation rate up to 85%, and clumping rate only 3.13%.

[0021] (2) High yield: The total number of cells obtained by dissociation reached 200,000, and the median number of genes reached 2382. Attached Figure Description

[0022] Figure 1 The image shows the analysis results of the cell counter in Example 1 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0023] Figure 2 The image shows the analysis results of the cell counter in Example 2 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0024] Figure 3 The image shows the analysis results of the cell counter in Example 3 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0025] Figure 4 The image shows the analysis results of the cell counter in Example 4 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0026] Figure 5 The image shows the analysis results of the cell counter in Example 5 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0027] Figure 6 The image shows the analysis results of the cell counter in Example 6 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0028] Figure 7The image shows the analysis results of the cell counter in Example 7 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0029] Figure 8 The image shows the analysis results of the cell counter in Example 8 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0030] Figure 9 The image shows the analysis results of the cell counter in Example 9 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0031] Figure 10 The image shows the analysis results of the cell counter in Example 10 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0032] Figure 11 The image shows the analysis results of the cell counter in Example 11 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0033] Figure 12 The image shows the analysis results of the cell counter in Example 12 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0034] Figure 13 The image shows the analysis results of the cell counter in Example 13 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0035] Figure 14The image shows the analysis results of the cell counter in Example 14 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right).

[0036] Figure 15 The image shows the analysis results of the cell counter in Example 15 of this application. The top row shows the fluorescently labeled images, from left to right: BR image, FL1 image, FL2 image and fusion image; the middle row shows the cell diameter distribution; and the bottom row shows the FL1 fluorescence intensity distribution (left) and FL2 fluorescence intensity distribution (right). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] As described above, in a first aspect, this application provides a single-cell extraction kit for cartilage tissue, the extraction kit comprising:

[0039] (1) The first enzyme for hydrolysis, wherein the first enzyme for hydrolysis includes one or more of trypsin, chondroitin ABC and fucosidase;

[0040] (2) The second enzyme used for hydrolysis includes collagenase II, neutral protease II, hyaluronidase and Dnase I (deoxyribonuclease I).

[0041] (3) Base solution, which includes base culture medium and / or buffer solution.

[0042] First enzymatic hydrolysis enzyme

[0043] In this invention, the first enzyme is mainly used to perform preliminary digestion of cartilage tissue, providing support for the second enzyme to perform enzymatic digestion more smoothly.

[0044] a. trypsin

[0045] In some preferred embodiments, the first enzymatic hydrolysate includes trypsin.

[0046] Trypsin is primarily used to hydrolyze cell surface proteins, further dissociating cells. While trypsin was used in early studies, it only achieved 65% cell viability. Subsequent mainstream single-cell extraction methods rarely use trypsin, mainly because it indiscriminately cleaves all membrane proteins, leading to a sharp drop in cell viability and excessive matrix degradation. However, our research has found that trypsin can still be used in specific scenarios, but its addition timing, concentration, and enzymatic hydrolysis time must be strictly controlled.

[0047] The inventors have discovered that, when using trypsin hydrolysis, trypsin hydrolysis can be used in the first hydrolysis stage with a trypsin solution at a working concentration of 0.05% to 0.1% (e.g., 0.05%, 0.06%; 0.07%, 0.08%, 0.09% or 1.00%), incubated at 37°C for 5 to 20 minutes (e.g., 10 minutes or 15 minutes) to loosen surface proteoglycans.

[0048] b. Chondroitinase ABC

[0049] In some preferred embodiments, the first enzymatic hydrolysate includes chondroitinase ABC.

[0050] Chondroitinase ABC can break down chondroitin sulfate, a major component of cartilage tissue, and should theoretically be well-suited for processing cartilage tissue. However, there are currently no reports of its use for single-cell extraction of cartilage tissue. This may be because chondroitinase ABC is generally believed to only process glycosaminoglycan side chains and is ineffective against the core collagen network. Furthermore, it is thought to potentially affect cell surface receptors such as CD44 or cell membrane integrity.

[0051] However, this invention has found that first using an appropriate concentration of chondroitinase ABC to enzymatically hydrolyze cartilage tissue can weaken the proteoglycan barrier, and then using collagenase II and other enzymes for enzymatic hydrolysis can significantly improve cell yield while ensuring cell viability.

[0052] When using chondroitin ABC for combined enzymatic hydrolysis, chondroitin ABC (e.g., purchased from Shanghai Guchen Biotechnology Co., Ltd.) can be prepared into a 10 U / mL stock solution using a basal solution. This solution should be prepared fresh and used immediately. The pH can be 7.2 to 8.0, for example, 7.4, consistent with physiological pH, making chondroitin ABC an ideal choice. The working concentration can be 0.1 U / mL to 1 U / mL, for example, 0.5 U / mL. The treatment temperature can be 37℃, and the treatment time can be 10 minutes to 60 minutes, for example, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes. When used in conjunction with trypsin, the time required for trypsin hydrolysis can be used.

[0053] The inventors have discovered that after the chondroitin ABC enzymatic hydrolysis is completed, centrifugation is not required before proceeding to the next enzymatic hydrolysis stage. Therefore, in this invention, if chondroitin ABC is used alone for the first enzymatic hydrolysis, the second enzymatic hydrolysis stage can be directly initiated.

[0054] c. Fucosidase

[0055] In some preferred embodiments, the first enzymatic hydrolysate includes fucosidase.

[0056] The single-cell extraction process of cartilage tissue has always focused on breaking down its robust proteoglycans and collagen network. Therefore, current technologies mainly focus on the research of traditional enzymes such as collagenase II, hyaluronidase, and Dnase I. There are no reports on the research and application of fucosidase in the single-cell extraction of cartilage tissue, which is speculated to be due to concerns about its impact on cell viability.

[0057] In their experiments, the inventors combined fucosidase with conventional enzymes and found significant improvements in cell yield and cell viability (possibly due to the ability of proteases to improve cell state). This is likely because many proteoglycans and glycoproteins in the cartilage cell matrix may have fucosylation on their glycan chains. These modifications may be involved in cell recognition, adhesion, or maintaining matrix structural stability. By hydrolyzing these fucosidic bonds, the following effects may be achieved: disrupting some sugar-mediated cell-matrix or cell-cell connections; loosening certain parts of the matrix, exposing more action sites, which facilitates the more effective penetration and degradation of collagen networks and proteoglycan polymers by major enzymes such as collagenase II and hyaluronidase; and exposing antigenic epitopes or protease cleavage sites that are shielded by glycan chains.

[0058] Furthermore, compared to some potent proteases such as trypsin, fucosidase is gentler, reducing damage to cell surface proteins and resulting in cells with higher activity and closer resemblance to their in vivo state. Therefore, fucosidase can be used in combination with traditional enzymes to improve cell yield; it can also replace or partially replace trypsin to better maintain cell state and viability.

[0059] Fucosidase is commercially available, for example from Beijing Bio-Lab Technology Co., Ltd., Megazyme, Sigma-Aldrich, or Shanghai Jinpan Biotechnology Co., Ltd.

[0060] The active temperature for fucosidase can be from 25°C to 40°C, preferably from 25°C to 37°C. The pH can be from 7.0 to 8.0, preferably 7.4. The digestion time can be from 10 minutes to 1 hour. When used in conjunction with trypsin, the digestion time required by trypsin can be used. The concentration used can be from 0.1 U / mL to 2.0 U / mL, for example from 0.5 U / mL to 1.0 U / mL.

[0061] Second enzymatic hydrolysis enzyme

[0062] The second enzymatic hydrolysis enzyme plays a major role in the enzymatic hydrolysis of cartilage tissue to release cells. In this invention, the second enzymatic hydrolysis enzyme simultaneously includes collagenase II, neutral protease II, hyaluronidase, and Dnase I. The working concentrations of these enzymes can be adjusted by those skilled in the art according to the actual enzymatic hydrolysis conditions.

[0063] In some preferred embodiments, the second enzyme for enzymatic hydrolysis may further include DNase I. The working concentration of DNase I in the second enzymatic hydrolysate may be from 0.05 mg / mL to 0.5 mg / mL, for example, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL or 0.5 mg / mL.

[0064] base fluid

[0065] The base solution used in this invention can be a culture medium or a buffer solution, preferably a culture medium.

[0066] In some preferred embodiments, the basal culture medium is DMEM medium, F12 medium, or RPMI 1640 medium. More preferably, the basal culture medium is RPMI 1640 medium.

[0067] Preferably, the buffer solution is PBS buffer (phosphate buffer).

[0068] auxiliary reagents

[0069] Auxiliary reagents are mainly added to the base solution for purposes such as pH buffering, antioxidant protection, or improving cell yield.

[0070] Therefore, in some preferred embodiments, the extraction kit further includes (4) auxiliary reagents, which include one or more of glycerol, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), BSA (Bovine Serum Albumin), FBS (Fetal Bovine Serum), and calcium chloride.

[0071] Glycerin, HEPES, BSA, FBS, and calcium chloride can be added to the base solution as components of the base solution. Thus, in some preferred embodiments, the base solution comprises one or more of glycerin, HEPES, BSA, FBS, and calcium chloride.

[0072] When the base solution contains glycerol, the concentration of glycerol is from 5% to 20% by volume (e.g., 5%, 10%, 15%, or 20% by volume). Glycerol is primarily used as a cryoprotectant to prevent enzyme inactivation at low temperatures.

[0073] When the base solution contains HEPES, the working concentration can be from 10 mM to 50 mM, for example, 10 mM, 20 mM, 30 mM, 40 mM or 50 mM. HEPES has a strong buffering capacity, which can more advantageously maintain the pH within the expected range, such as 7.2 to 7.4, avoiding enzyme denaturation caused by acidic or alkaline environments, and is particularly suitable for steps with long enzymatic hydrolysis times.

[0074] When the base solution contains BSA (bovine serum albumin), the working concentration of the BSA can be from 0.1 wt / volume% to 1 wt / volume%, for example, 0.1 wt / volume%, 0.2 wt / volume%, 0.3 wt / volume%, 0.4 wt / volume%, 0.5 wt / volume%, 0.6 wt / volume%, 0.7 wt / volume%, 0.8 wt / volume%, 0.9 wt / volume%, or 1.0 wt / volume.

[0075] In this invention, the core function of BSA is to reduce cell adhesion. Freshly separated cells have very sticky surfaces, easily aggregating or adhering to centrifuge tube walls, pipette tips, etc., leading to cell loss. BSA can pre-coat these surfaces, providing a non-sticky coating, thereby improving cell yield. Another function of BSA is its carrier and stabilizing effect: BSA can bind and transport hydrophobic molecules such as fatty acids and hormones, stabilizing these beneficial components for cells. It can also bind some toxic metabolites or heavy metal ions, playing a role in detoxification and cell protection. Yet another function of BSA is to provide osmotic pressure support: as a macromolecule, BSA can help maintain the osmotic pressure balance of the solution. BSA also reduces protein adsorption and denaturation on tube walls.

[0076] One function of FBS is to provide nutrients such as amino acids, vitamins, and growth factors, offering nutritional support to newly separated, "traumatized" cells. Another function of FBS is to reduce mechanical damage: during operations such as pipetting and centrifugation, it can form a protective layer around the cells, reducing the damage caused by mechanical shear forces. In the basal solution, the working concentration of FBS can range from 0% by weight (w / v) to 10% by weight (w / v).

[0077] Calcium chloride plays a role in the single-cell extraction of cartilage tissue mainly by maintaining the extracellular calcium ion concentration, regulating cell metabolism and function, and preventing cell aggregation, thereby improving the efficiency and quality of single-cell extraction. When the base solution contains calcium chloride, its concentration in the base solution can be from 1 mM to 10 mM, for example, 1 mM, 2 mM, 5 mM or 10 mM.

[0078] The present invention also provides a single-cell extraction kit for cartilage tissue, the extraction kit comprising a first enzymatic hydrolysate prepared from the first enzymatic hydrolysate and the base solution described above; and a second enzymatic hydrolysate prepared from the second enzymatic hydrolysate and the base solution described above. The concentrations of various enzymes in the first and second enzymatic hydrolysates can be independently set to a working concentration of 1 to 10 times (e.g., 1, 2, 5, or 10 times).

[0079] In some embodiments, the various enzymes in the first and second enzymes included in the extraction kit may each be in the form of a stock solution with a working concentration of 1 to 10 times (e.g., 1, 2, 5, or 10 times), or in the form of a mixed stock solution containing two or more enzymes with a working concentration of 1 to 10 times (e.g., 1, 2, 5, or 10 times).

[0080] Preferably, the extraction kit may further include a base solution for diluting the mother solution or washing tissue, tube walls, or precipitate, the base solution may or may not contain one or more of the aforementioned auxiliary reagents. If one or more of the aforementioned auxiliary reagents are included, the base solution may contain each auxiliary reagent at a working concentration.

[0081] The working concentrations of the enzymes or auxiliary reagents mentioned in this regard will be discussed elsewhere in the context and will not be repeated here.

[0082] In a second aspect, the present invention provides a method for extracting single cells from cartilage tissue, wherein the extraction method uses the extraction kit described in the first aspect of the present invention.

[0083] In some preferred embodiments, the method includes the following steps:

[0084] (1) Tissue fragmentation: The cartilage tissue is cut into fragments.

[0085] (2) First stage of enzymatic hydrolysis: The cartilage tissue fragments are enzymatically hydrolyzed using the first enzymatic hydrolysis solution prepared by the first enzymatic hydrolysis enzyme and the base solution, and then centrifuged, the supernatant is discarded, and the first enzymatic hydrolysis material is obtained.

[0086] (3) Second stage of enzymatic hydrolysis: The first enzymatic hydrolysate is enzymatically hydrolyzed using a second enzymatic hydrolysate prepared from a second enzymatic hydrolysate and a base solution, and then filtered using a mesh sieve to obtain the filtrate;

[0087] (4) Centrifuge the filtrate and collect the precipitate as the target single cell.

[0088] Preferably, in step (1), the cartilage tissue is cut to a size no larger than 1 mm. 3 Fragments, for example, 0.8 mm 3 .

[0089] Preferably, in step (1), the cartilage tissue can be rinsed three times with PBS buffer and then cut into fragments smaller than 1 cubic millimeter.

[0090] Preferably, in the first enzymatic hydrolysate of step (2), the concentration of the trypsin is from 0.05 wt% to 0.25 wt% or 0.1 wt% or 0.15 wt% or 0.20 wt% or 0.20 wt%.

[0091] Preferably, in the first enzymatic hydrolysate of step (2), the concentration of chondroitin ABC is 0.1 U / mL to 1 U / mL, for example 0.2 U / mL or 0.5 U / mL.

[0092] Preferably, in the first enzymatic hydrolysate of step (2), the concentration of the fucosidase is from 0.1 U / mL to 2.0 U / mL, for example, 0.2 U / mL, 0.5 U / mL, or 1.0 U / mL.

[0093] In some preferred embodiments, the concentration of collagenase II in the second enzymatic hydrolysate in step (3) is from 0.05 wt% to 0.2 wt% to 0.1 wt% or 0.1 ...

[0094] Preferably, in the second enzymatic hydrolysate of step (3), the concentration of neutral proteinase II is from 0.05 wt% to 0.3 wt% or 0.20 wt% or 0.25 wt% or 0.1 wt% or 0.15 wt% or 0.20 wt% or 0.25 wt% or 0.25 wt%.

[0095] Preferably, the concentration of the hyaluronidase is from 0.1 U / mL to 0.5 U / mL, for example, 0.2 U / mL, 0.3 U / mL or 0.4 U / mL.

[0096] Preferably, the sieve filtration is performed using a 70μm mesh sieve.

[0097] Preferably, the centrifugation mentioned in each step of the method of the present invention can be independently defined as centrifugation at 300g for 5 minutes at 4°C. Example

[0098] The present invention will be further described below through embodiments. These embodiments are for illustrative purposes only, and the scope of protection of the present invention is not limited to these embodiments.

[0099] Unless otherwise specified, the reagents and instruments used in the examples are all known and commercially available.

[0100] The main preparation reagents used in the examples are as follows:

[0101] (1) Base fluid

[0102] A basal solution containing 10 v / v glycerol, 20 mM HEPES, 0.5 w / v % BSA, 5 w / v FBS and 5 mM calcium chloride was prepared using RPMI 1640 medium as the basal medium.

[0103] (2) First enzyme hydrolysate mother liquor (10 times concentration)

[0104] The first enzymatic hydrolysate containing trypsin, chondroitin ABC and / or fucosidase was prepared using the base solution obtained in the first enzymatic hydrolysis (1).

[0105] In use, the base solution prepared in (1) is used to dilute the first enzymatic hydrolysate mother liquor by 10 times, so that each enzyme used in the first enzymatic hydrolysis is diluted to the following concentrations: the concentration of trypsin is 0.1 weight / volume; the concentration of chondroitin ABC is 0.5 U / mL, and the concentration of fucosidase is 0.5 U / mL.

[0106] (3) Second enzyme hydrolysate mother liquor (10 times concentration)

[0107] A second enzymatic hydrolysate containing collagenase II, neutral protease II, hyaluronidase and Dnase I was prepared using the second enzymatic hydrolysate and the base solution obtained in (1).

[0108] In use, the base solution prepared in (1) is used to dilute the second enzymatic hydrolysate mother liquor by 10 times, so that each second enzymatic hydrolysate enzyme is diluted to the following concentrations to obtain the second enzymatic hydrolysate: the working concentration of collagenase II is 0.1 wt / volume%, the working concentration of neutral proteinase II is 0.2 wt / volume%, the working concentration of hyaluronidase is 0.2 U / mL, and the working concentration of Dnase I is 0.1 mg / mL.

[0109] (5) Screen

[0110] The filtration process uses a 70-micron nylon mesh screen.

[0111] Unless otherwise specified, the pH of each liquid reagent is 7.4.

[0112] Example 1

[0113] In this embodiment, the first enzymatic hydrolysate mother liquor contains only trypsin as the first enzymatic hydrolysate.

[0114] The specific steps for extraction are as follows:

[0115] 1. Take a 5mL centrifuge tube and add 3mL of the first dissociation solution prepared from the first enzyme hydrolysate mother liquor and the base solution.

[0116] 2. Wash the mouse articular cartilage tissue three times with PBS buffer (pH 7.4), weigh 0.5g of mouse articular cartilage tissue and cut it into pieces no larger than 1mm. 3 Remove the fragments and transfer them to the solution prepared in step 1, then shake to mix.

[0117] 3. Place in a 37℃ water bath for 20 minutes, inverting and mixing every 3-5 minutes during the process.

[0118] 4. After digestion, centrifuge at 300g for 5 minutes at 4℃ and discard the supernatant.

[0119] 5. Add 3 mL of basal culture medium to the precipitate, mix well by pipetting, centrifuge at 300×g for 5 min at 4℃, and discard the supernatant.

[0120] 6. Add 2720 μL of basal culture medium and 280 μL of the second enzyme digest to the precipitate, mix well by pipetting, and place in a 37℃ water bath for 10 h (6 h to 12 h). During this period, invert and mix at intervals.

[0121] 7. After digestion, filter the solution through a 70 μm cell sieve and collect the filtrate in a new 15 mL centrifuge tube.

[0122] 8. Add 3 mL of basal culture medium to the centrifuge tube used for digestion in step 6 and invert it to rinse the tube wall to obtain the rinsing solution. Filter the rinsing solution through the 70 μm cell sieve used in step 7 and collect the filtrate to combine it into the 15 mL centrifuge tube used in step 7.

[0123] 9. Repeat step 8 twice, that is, wash a total of 3 times, combine the filtrates, and obtain the collected liquid (about 12 mL).

[0124] 10. Centrifuge the collected liquid at 300×g for 5 min at 4℃ and discard the supernatant.

[0125] 11. Resuspend the precipitate in 5 mL of PBS buffer containing 5% FBS, mix well by pipetting, centrifuge at 300×g for 5 min at 4℃, and discard the supernatant.

[0126] 12. Repeat step 11 once.

[0127] 13. Resuspend the pellet in 100 μL (or 50 μL to 2 mL if necessary) of PBS buffer containing 5% FBS (adjust the volume of the resuspending solution according to the desired cell concentration to obtain the required single-cell suspension).

[0128] 14. Perform the following tests on the cell suspension obtained in step 13:

[0129] (1) Cell viability detection: Countstar RE010212 AO / PI dye (Shanghai Ruiyu Biotechnology Co., Ltd. RE010212) was used.

[0130] (2) Cell viability, clumping rate, nucleation rate and cell number: were determined using a Countstar cell counter.

[0131] (3) Detection of gene median:

[0132] Single-cell sequencing experiments were performed on the prepared single-cell suspension using 10×Genomics technology. Cell capture, water-in-oil emulsion generation, and subsequent library construction were performed according to the standard procedure provided by 10×Genomics.

[0133] Sequencing results are shown in Table 1 and Figure 1 As shown, the cell viability was 88.95%, the viable cell concentration was 1.2E+06 / mL, the cell clumping rate was 5.93%, the nucleation rate was 79%, and the total number of viable cells was approximately 160,000. The average cell diameter was 9.75 μm, and the average roundness was 0.93. The fluorescence spectrum and distribution map analyzed by the cell counter in this embodiment are shown below. Figure 1 As shown; the median of genes is shown in Table 1 separately (the same applies to Examples 2 to 15, so they will not be described one by one).

[0134] Example 2

[0135] The process was carried out in basically the same manner as in Example 1, except that the first enzymatic hydrolysate mother liquor used in this example contained only chondroitin ABC as the first enzymatic hydrolysate.

[0136] Sequencing results are shown in Table 1 and Figure 2 As shown, the cell viability was 87.72%, the viable cell concentration was 1.0E+06 / mL, the cell clumping rate was 2.45%, the nucleation rate was 80%, and the total number of viable cells was approximately 150,000. The average cell diameter was 9.76 μm, and the average roundness was 0.82.

[0137] Example 3

[0138] The process was carried out in basically the same manner as in Example 1, except that the first enzymatic hydrolysate mother liquor used in this example contained only fucosidase as the first enzymatic hydrolysate.

[0139] Sequencing results are shown in Table 1 and Figure 3 As shown, the cell viability was 82.66%, the viable cell concentration was 8.94E+05 / mL, the cell clumping rate was 4.54%, the nucleation rate was 78%, and the total number of viable cells was approximately 140,000. The average cell diameter was 10.53 μm, and the average roundness was 0.95.

[0140] Example 4

[0141] The process was carried out in basically the same manner as in Example 1, except that the first enzymatic hydrolysate mother liquor used in this example contained trypsin and chondroitin ABC as the first enzymatic hydrolysate.

[0142] Sequencing results are shown in Table 1 and Figure 4 As shown, the cell viability was 93.34%, the viable cell concentration was 1.01E+06 / mL, the cell clumping rate was 3.13%, the nucleation rate was 85%, and the total number of viable cells was approximately 200,000. The average cell diameter was 10.95 μm, and the average roundness was 0.94.

[0143] Example 5

[0144] The process was carried out in basically the same manner as in Example 1, except that the first enzymatic hydrolysate mother liquor used in this example contained trypsin and fucosidase as the first enzymatic hydrolysate enzymes.

[0145] Sequencing results are shown in Table 1 and Figure 5 As shown, the cell viability was 92.6%, the viable cell concentration was 1.03E+06 / mL, the cell clumping rate was 4.25%, the nucleation rate was 86%, and the total number of viable cells was approximately 190,000. The average cell diameter was 10.83 μm, and the average sphericity was 0.94.

[0146] Example 6

[0147] The process was carried out in basically the same manner as in Example 1, except that the first enzymatic hydrolysate mother liquor used in this example contained chondroitinase ABC and fucosidase as the first enzymatic hydrolysate.

[0148] Sequencing results are shown in Table 1 and Figure 6 As shown, the cell viability was 91.66%, the viable cell concentration was 1.13E+06 / mL, the cell clumping rate was 3.94%, the nucleation rate was 82%, and the total number of viable cells was approximately 190,000. The average cell diameter was 10.02 μm, and the average roundness was 0.93.

[0149] Example 7

[0150] The process was carried out in basically the same manner as in Example 1, except that the first enzymatic hydrolysate mother liquor used in this example contained trypsin, chondroitin ABC and fucosidase as the first enzymatic hydrolysate.

[0151] Sequencing results are shown in Table 1 and Figure 7 As shown, the cell viability was 84.2%, the viable cell concentration was 1.2E+06 / mL, the cell clumping rate was 4.28%, the nucleation rate was 76%, and the total number of viable cells was approximately 140,000. The average cell diameter was 8.92 μm, and the average sphericity was 0.94.

[0152] Example 8

[0153] The procedure was carried out in essentially the same manner as in Example 1, except that the trypsin used as the first enzyme for hydrolysis was added directly to the second hydrolysate instead of the first hydrolysate. In other words, the first hydrolysate used in this example did not contain any first enzyme for hydrolysis. In other words, trypsin was used in combination with collagenase II, neutral protease II, hyaluronidase and Dnase I in a one-step process.

[0154] Sequencing results are shown in Table 1 and Figure 8 As shown, the cell viability was 77.43%, the viable cell concentration was 1.2E+06 / mL, the cell clumping rate was 5.98%, the nucleation rate was 75%, and the total number of viable cells was approximately 70,000. The average cell diameter was 10.96 μm, and the average roundness was 0.95.

[0155] Example 9

[0156] The procedure was carried out in essentially the same manner as in Example 2, except that chondroitin ABC, which was used as the first enzyme for hydrolysis, was added directly to the second hydrolysate instead of the first hydrolysate. In other words, the first hydrolysate used in this example did not contain any first enzyme for hydrolysis. In other words, chondroitin ABC was used in a one-step process in combination with collagenase II, neutral protease II, hyaluronidase and Dnase I.

[0157] Sequencing results are shown in Table 1 and Figure 9 As shown, the cell viability was 75.15%, the viable cell concentration was 8.99E+05 / mL, the cell clumping rate was 7.53%, the nucleation rate was 73%, and the total number of viable cells was approximately 60,000. The average cell diameter was 10.91 μm, and the average roundness was 0.97.

[0158] Example 10

[0159] The procedure was carried out in essentially the same manner as in Example 3, except that the fucosidase used as the first enzymatic hydrolysis enzyme was added directly to the second enzymatic hydrolysate instead of the first enzymatic hydrolysate. In other words, the first enzymatic hydrolysate used in this example did not contain any first enzymatic hydrolysis enzyme. In other words, a one-step method was used to combine fucosidase with collagenase II, neutral protease II, hyaluronidase and Dnase I.

[0160] Sequencing results are shown in Table 1 and Figure 10 As shown, the cell viability was 71.51%, the viable cell concentration was 9.8E+05 / mL, the cell clumping rate was 6.89%, the nucleation rate was 68%, and the total number of viable cells was approximately 50,000. The average cell diameter was 10.17 μm, and the average roundness was 0.95.

[0161] Example 11

[0162] The process was carried out in basically the same manner as in Example 1, except that the first enzymatic hydrolysate mother liquor used in this example did not contain the first enzymatic hydrolysate enzyme.

[0163] Sequencing results are shown in Table 1 and Figure 11 As shown, the cell viability was 73.45%, the viable cell concentration was 1.15E+06 / mL, the cell clumping rate was 8.55%, the nucleation rate was 70%, and the total number of viable cells was approximately 40,000. The average cell diameter was 10.56 μm, and the average roundness was 0.89.

[0164] Example 12

[0165] The procedure was carried out in essentially the same manner as in Example 4, except that HBSS containing 1 g / mL ascorbic acid was used instead of the PBS buffer used in each step of Example 1.

[0166] Sequencing results are shown in Table 1 and Figure 12 As shown, the cell viability was 90.52%, the viable cell concentration was 1.1E+06 / mL, the cell clumping rate was 5.06%, the nucleation rate was 83%, and the total number of viable cells was approximately 180,000. The average cell diameter was 10.59 μm, and the average roundness was 0.93.

[0167] Example 13

[0168] The procedure was carried out in essentially the same manner as in Example 4, except that DMEM medium was used instead of the RPMI 1640 medium used in Example 1 to prepare the reagents.

[0169] Sequencing results are shown in Table 1 and Figure 13 As shown, the cell viability was 91.59%, the viable cell concentration was 1.03E+06 / mL, the cell clumping rate was 6.45%, the nucleation rate was 82%, and the total number of viable cells was approximately 200,000. The average cell diameter was 10.66 μm, and the average roundness was 0.96.

[0170] Example 14

[0171] The procedure was carried out in essentially the same manner as in Example 4, except that the working concentrations of the first and second enzymes for enzymatic hydrolysis were adjusted as follows:

[0172] The concentration of trypsin was 0.05% by weight / volume%.

[0173] The concentration of chondroitin ABC was 0.1 U / mL;

[0174] The concentration of fucosidase was 0.1 U / mL;

[0175] The concentration of collagenase II was 0.05% by weight / volume%.

[0176] The concentration of neutral proteinase II was 0.05% by weight / volume.

[0177] The concentration of hyaluronidase was 0.1 U / mL.

[0178] Sequencing results are shown in Table 1 and Figure 14 As shown, the cell viability was 88.73%, the viable cell concentration was 1.21E+06 / mL, the cell clumping rate was 7.51%, the nucleation rate was 81%, and the total number of viable cells was approximately 160,000. The average cell diameter was 8.8 μm, and the average roundness was 0.95.

[0179] Example 15

[0180] The procedure was carried out in essentially the same manner as in Example 4, except that the working concentrations of the first and second enzymes for enzymatic hydrolysis were adjusted as follows:

[0181] The concentration of trypsin was 0.25% by weight / volume.

[0182] The concentration of chondroitin ABC is 1 U / mL;

[0183] The concentration of fucosidase was 2.0 U / mL;

[0184] The concentration of collagenase II was 0.2% by weight / volume.

[0185] The concentration of neutral proteinase II was 0.3% by weight / volume.

[0186] The concentration of hyaluronidase was 0.5 U / mL.

[0187] Sequencing results are shown in Table 1 and Figure 15 As shown, the cell viability was 90.24%, the viable cell concentration was 1.19E+06 / mL, the cell clumping rate was 5.81%, the nucleation rate was 82%, and the total number of viable cells was approximately 180,000. The average cell diameter was 8.91 μm, and the average roundness was 0.94.

[0188] Table 1. Quality indicators of single-cell products obtained in each example

[0189]

[0190] The results from the various embodiments show that the various performance indicators (including cell viability, cell clumping rate, and nucleation rate) of Examples 1-7 and Examples 12-15 all meet the requirements for subsequent experiments. Examples 2 (using chondroitin ABC as the first enzyme) and 3 (using fucosidase as the first enzyme) achieved technical effects comparable to Example 1 (using trypsin as the first enzyme). Examples 4-6, using two enzymes selected from trypsin, chondroitin ABC, and fucosidase as the first enzyme, achieved cell viability of over 90%. Example 4, using a first enzyme digest containing both trypsin and chondroitin ABC as the first enzyme, yielded the best results, even surpassing Example 7, which used all three enzymes: trypsin, chondroitin ABC, and fucosidase. Examples 8-11, by simultaneously adding the first and second enzymes, significantly affected cell viability. Examples 12-15 also achieved relatively ideal results.

[0191] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting single cells from cartilage tissue, characterized in that: The extraction method used a single-cell extraction kit for cartilage tissue. The single-cell extraction kit for cartilage tissue includes: (1) a first enzyme for digestion, which is composed of two of trypsin, chondroitin ABC and fucosidase; (2) a second enzyme for digestion, which includes collagenase II, neutral protease II, hyaluronidase and dnase I; and (3) a basal solution, which includes basal culture medium and / or buffer. The method includes the following steps: (1) Tissue fragmentation: The cartilage tissue is cut into fragments; (2) First stage of enzymatic hydrolysis: The fragments are enzymatically hydrolyzed using the first enzymatic hydrolysate prepared from the first enzymatic hydrolysate and the base solution, then centrifuged, and the supernatant is discarded to obtain the first enzymatic hydrolysate material; (3) Second stage of enzymatic hydrolysis: The first enzymatic hydrolysate is enzymatically hydrolyzed using the second enzymatic hydrolysate prepared from the second enzymatic hydrolysate and the base solution, and then filtered using a mesh sieve to obtain the filtrate; (4) Centrifuge the filtrate and collect the precipitate as the target single cell; In the first enzymatic hydrolysate in step (2): the concentration of trypsin is 0.05 wt% to 0.25 wt%; the concentration of chondroitin ABC is 0.1 U / mL to 1 U / mL; and / or the concentration of fucosidase is 0.1 U / mL to 2.0 U / mL; In the second enzymatic hydrolysate in step (3): the concentration of collagenase II is 0.05 wt% to 0.2 wt%; the concentration of neutral protease II is 0.05 wt% to 0.3 wt%; the concentration of hyaluronidase is 0.1 U / mL to 0.5 U / mL; and / or the concentration of Dnase I is 0.05 mg / mL to 0.5 mg / mL.

2. The method according to claim 1, characterized in that, The extraction kit also includes: (4) Auxiliary reagents, wherein the auxiliary reagents include one or more of glycerol, HEPES, BSA, FBS and calcium chloride.

3. The method according to claim 1, characterized in that, The basal culture medium is DMEM medium, F12 medium or RPMI 1640 medium.

4. The method according to claim 1, characterized in that, The buffer solution is PBS buffer.

5. The method according to claim 1, characterized in that, In step (1), the cartilage tissue is cut to a size no larger than 1 mm. 3 Fragments.

6. The method according to claim 1, characterized in that, The base solution also contains one or more auxiliary reagents, and the working concentrations of each auxiliary reagent are as follows: The concentration of glycerol is 5% to 20% by volume. The concentration of HEPES ranges from 10 mM to 50 mM; The concentration of BSA ranges from 0.1 wt% to 1 wt%. The concentration of FBS is from 0 wt% to 10 wt%; and / or The concentration of calcium chloride is 1 mM to 10 mM.

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