A kit and method for preparing cells of the class reptilia

Abstract

This invention discloses a kit and method for preparing single cells from reptiles, belonging to the field of single-cell sequencing technology. The kit includes Collagenase I, DNase I, Dispase, and DMEM culture medium. The kit and method of this invention fill the gap in single-cell preparation technology from reptile tissues, especially the Chinese alligator. The kit has simple components and is easy to prepare; the method has high reproducibility and success rate, and can obtain large quantities of single cells with high viability and low impurities, which can be used for single-cell sequencing and other applications.

Description

Technical Field

[0001] This invention belongs to the field of single-cell sequencing technology, specifically, it relates to a kit and method for preparing reptile single cells, and more specifically, it relates to a kit and method for preparing Chinese alligator single cells. Background Technology

[0002] Single-cell transcriptome sequencing is a new technology that sequences the transcriptome at the single-cell level. It can study gene expression within a single cell and solve the problem of cellular heterogeneity that conventional transcriptome sequencing cannot address, making it possible to analyze the behavior, mechanisms, and relationship of individual cells with the organism. In recent years, this technology has been widely applied to various tissues of many species, but mainly to mammals (especially humans and mice).

[0003] Currently, there are still many challenges in conducting single-cell transcriptome sequencing on other species: most single-cell preparation methods are based on mammals (such as humans and mice), and are not well-suited for tissues of animal groups such as reptiles that are not closely related to mammals. Single-cell preparation techniques for homologous tissues from different species cannot be used directly.

[0004] The Chinese alligator is a species of crocodile endemic to my country. Currently, its survival status is critically endangered. Research and conservation efforts for the Chinese alligator are extremely urgent. However, there is currently no single-cell preparation method for Chinese alligator-related tissues. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention aims to provide a single-cell preparation method for the gonads of Yangtze alligator embryos. To achieve this objective, the technical solution adopted by this invention is as follows:

[0006] The first aspect of the present invention provides a kit for preparing single cells of reptiles, the kit comprising Collagenase I, DNase I, Dispase and DMEM culture medium.

[0007] In some embodiments of the present invention, the DMEM culture medium contains 3-8% FBS.

[0008] In some embodiments of the present invention, Collagenase I and DNase I are pre-added to DMEM culture medium at final concentrations of 0.5–2 mg / mL and 0.1–1 mg / mL, respectively. In some preferred embodiments of the present invention, the final concentrations are 1 mg / mL and 0.5 mg / mL, respectively.

[0009] A second aspect of the present invention provides a method for preparing reptile single cells using the kit described in the first aspect of the present invention, comprising the following steps:

[0010] S1, Collagenase I and DNase I are pre-added to DMEM medium at final concentrations of 0.5–2 mg / mL and 0.1–1 mg / mL, respectively, preferably 1 mg / mL and 0.5 mg / mL, respectively;

[0011] S2, Obtain tissue samples of the reptile and cut them into 1mm pieces. 3 The small pieces were transferred to the DMEM medium containing Collagenase I and DNase I obtained in step S1;

[0012] S3, transfer to a constant temperature shaker, 30-35℃, 60-100rpm for 5-20min;

[0013] S4, remove and mix well, add 0.5 mg / mL Dispase, return to the constant temperature shaker, and digest for 10-30 min;

[0014] S5. Pass the culture medium through a 30μm sieve, collect the supernatant, centrifuge at 280-400g for 3-10 minutes at 8-15℃, discard the supernatant, and resuspend the precipitate in buffer to obtain the first cell suspension.

[0015] In some embodiments of the present invention, the method further includes:

[0016] S6. Wash off the remaining tissue on the sieve with an appropriate amount of DMEM medium, add 2 times the volume of 0.25% trypsin, shake at 30-35℃ and 60-100 rpm for 5-20 min, and repeat step S5 to obtain the second cell suspension.

[0017] In some embodiments of the invention, between steps S3 and S4 and / or between steps S4 and S5, a further step of removing and mixing the culture medium is included. In some embodiments of the invention, the culture medium is mixed by pipetting.

[0018] In some embodiments of the present invention, in step S5, the buffer solution is selected from the group consisting of PBS buffer, MOPS buffer, MES buffer, HEPES buffer, and Tris-HCl buffer. Preferably, the buffer solution is PBS buffer, and more preferably, the PBS buffer solution contains 5% FBS.

[0019] In some embodiments of the present invention, the reptile is the Chinese alligator. In some embodiments of the present invention, the tissue is embryonic gonadal tissue.

[0020] Beneficial effects of the present invention

[0021] Compared with the prior art, the present invention achieves the following beneficial effects:

[0022] The kit and method of this invention fill the gap in single-cell preparation technology from reptile tissues, especially the Chinese alligator. The kit has simple components and is easy to prepare, and the method has high reproducibility and success rate, enabling the production of large quantities of single cells with high viability and low impurities, which can be used for single-cell sequencing and many other applications. Attached Figure Description

[0023] Figure 1 The cell counting results of cell suspension A prepared in Example 2 of the present invention are shown.

[0024] Figure 2 The cell counting results of cell suspension B prepared in Example 2 of the present invention are shown.

[0025] Figure 3 The cell counting results after mixing cell suspension A and cell suspension B prepared in Example 2 of the present invention are shown.

[0026] Figure 4 The sequencing results of single-cell sequencing of cells prepared in Example 2 of the present invention are shown. Detailed Implementation

[0027] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, in particular the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0028] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. A numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly described in this application.

[0029] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0030] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0031] Example

[0032] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and referenced by them are incorporated herein by reference.

[0034] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0035] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed according to the specific methods listed in *Molecular Cloning: A Laboratory Manual* (4th Edition) (J. Sambrook & MR. Green, 2017), or according to the kit and product instructions. Other experimental methods, unless otherwise specified, were conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples were standard laboratory equipment; the experimental materials used in the following examples, unless otherwise specified, were purchased from a regular biochemical reagent store.

[0036] Example 1: Single-cell preparation reagents

[0037] This embodiment provides a reagent combination suitable for the preparation of single cells from reptile tissues, including Collagenase I, DNase I, Dispase, and DMEM culture medium (containing 5% FBS).

[0038] Before use, add Collagenase I and DNase I to DMEM medium to final concentrations of 1 mg / mL and 0.5 mg / mL, respectively, and keep on ice. Dispase is added during digestion (final concentration 0.5 mg / mL) to ensure cell viability and digestion efficiency.

[0039] Example 2: Single-cell preparation of gonadal tissue from Yangtze alligator embryos

[0040] The preparation of single cells from the gonadal tissue of Chinese alligator embryos using the reagent combination described in the examples is as follows:

[0041] a) Use PBS to wash the gonadal tissue of Yangtze alligator embryos;

[0042] b) Use a needle to cut the tissue into uniform 1mm pieces. 3 Small pieces on the left and right;

[0043] c) Transfer the tissue to the pre-cooled DMEM medium containing Collagenase I and DNase I prepared in Example 1, and incubate on ice for 10 min to allow the digestion solution to fully wet the tissue, improve dissociation efficiency, reduce dissociation time, and ensure cell viability;

[0044] d) Transfer to a constant temperature shaker, set the temperature to 35℃ (crocodile cells grow better at this temperature, the temperature conditions for crocodile survival are all below 35℃, and enzyme activity is almost unaffected under this condition, so it is a better choice), 80 rpm, 10 min;

[0045] e) Remove and blow-dry the food, then return it to the constant temperature shaker to continue digestion for 15 minutes;

[0046] f) Remove the sample, blow it into the container, add 0.5 mg / mL Dispase, return it to the constant temperature shaker, and digest for 15 min;

[0047] g) Remove and blow on it, observe the digestion, and continue digestion for 5-10 minutes;

[0048] h) Pass the sample through a 30μm sieve, collect the supernatant, centrifuge at 12℃ and 300g for 6 min, discard the supernatant, and resuspend the precipitate in 1.5mL PBS buffer (containing 5% FBS). This is called cell suspension A (the resuspension volume depends on the amount of precipitate and should be relatively small. Higher suspension concentrations can be diluted according to the counting concentration to avoid unnecessary operations).

[0049] i) At the same time, wash off the remaining tissue on the sieve with 1 mL of DMEM, add 2 mL of 0.25% trypsin, and continue digestion for 10 min in a constant temperature shaker at 35°C and 80 rpm;

[0050] j) Remove the sample, pipette it through a 30μm sieve, collect the supernatant, centrifuge at 12℃ and 300g for 6 min, discard the supernatant, and resuspend the precipitate in 1.5mL PBS buffer (containing 5% FBS), and label it cell suspension B.

[0051] The cell suspension prepared using the above method causes less damage to the cells, contains very few impurities, and has a high cell viability.

[0052] Cell suspension A is the dissociation product of mixed enzyme digestion. Its viability is in the range of 75%–90%, and it contains a small amount of impurities, such as… Figure 1 As shown, further cleaning and sieving through a 20μm sieve are required before use.

[0053] Cell suspension B is the product of further trypsin digestion of the remaining tissue after dissociation by mixed enzyme digestion solution. The suspension is of high quality, with a viability often exceeding 90% and few fragments. Figure 2 As shown, no further processing is required.

[0054] Next, the inventors used a cell counter to count the number of cells. To obtain complete cell types, cell suspensions A and B were mixed each time the cells were counted. The counting results are as follows: Figure 3 As shown, from Figure 3 The cell concentration and cell viability can be observed. The results of multiple counts are shown in Table 1.

[0055] Table 1 Single cell count

[0056]

[0057] Example 3 Single-cell sequencing

[0058] Single-cell sequencing was performed using the single cells prepared in Example 2. Single-cell RNA sequencing is a new technology that sequences the transcriptome at the single-cell level. It can study gene expression in a single cell and solve the problem of cellular heterogeneity that cannot be solved by sequencing tissue samples, making it possible to analyze the behavior, mechanisms and relationship of a single cell with the body.

[0059] The results are as follows Figure 4 As shown, from Figure 4 The results show that the cell expression level is high, the reproducibility is good, the proportion of mitochondria is low, and the data results are good.

[0060] Example 4: Performance Verification of Single-Cell Preparation Reagents

[0061] To verify the performance of the single-cell preparation reagent in Example 1, the inventors replaced one of the components or adjusted the content of one of the components, and prepared single cells of Chinese alligator embryonic gonadal tissue using the method described in Example 2. The results are shown in Table 2:

[0062] Table 2. Counting results of cell suspensions from different single-cell preparation reagents.

[0063]

[0064] As shown in Table 2, no substance in the single-cell preparation reagent in Example 1 can be arbitrarily replaced, and the concentration cannot be arbitrarily adjusted; otherwise, the concentration and viability of the prepared single cells will be significantly affected.

[0065] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing single cells of reptiles, characterized in that, Includes the following steps: S1, Collagenase I and DNase I were pre-added to DMEM medium to final concentrations of 1 mg / mL and 0.5 mg / mL, respectively; S2, Obtain tissue samples of the reptile and cut them into 1mm pieces. 3 The small pieces were transferred to the DMEM medium containing Collagenase I and DNase I obtained in step S1; S3, transfer to a constant temperature shaker, 30~35℃, 60~100rpm for 5~20min; S4, remove and mix well, add Dispase to a final concentration of 0.5 mg / mL, return to the constant temperature shaker, and digest for 10-30 min; S5. Pass the culture medium through a 30μm sieve, collect the supernatant, centrifuge at 8~15℃ and 280~400g for 3~10min, discard the supernatant, resuspend the precipitate in buffer solution to obtain the first cell suspension; S6: Wash off the remaining tissue on the sieve with an appropriate amount of DMEM medium, add 2 volumes of 0.25% trypsin, incubate at 30-35℃ and 60-100 rpm for 5-20 minutes, repeat step S5 to obtain the second cell suspension. Mix the first cell suspension and the second cell suspension. The reptile in question is the Chinese alligator, and the tissue is embryonic gonadal tissue.

2. The method according to claim 1, characterized in that, Between steps S3 and S4 and / or between steps S4 and S5, a further step of removing and mixing the culture medium is included.

3. The method according to claim 2, characterized in that, Use a pipette to mix the culture medium by blowing and blowing.

4. The method according to claim 1, characterized in that, In step S5, the buffer solution is selected from one of the group consisting of PBS buffer, MOPS buffer, MES buffer, HEPES buffer, and Tris-HCl buffer.

Images