Solution for separating nucleoplasm of adipocytes and application thereof

Abstract

The application discloses a solution for separating fat cell nucleus and cytoplasm, and the solution comprises solution 1 and solution 2; wherein the solution 1 comprises 8-12 mM hydrochloride buffer, 0.03-0.15% Tween-20, 8-12 mM sodium chloride, 2-4 mM magnesium chloride, 0.8-2% bovine serum albumin and 0.4-0.6% ethyl phenyl polyethylene glycol; the solution 2 comprises 8-12 mM hydrochloride buffer, 8-25 mM sodium chloride, 1-15 mM magnesium chloride and 0.8-2.5% bovine serum albumin; the percentage is volume percentage. The application can realize the research on all cell types of fat tissues, greatly reduces the required amount of fat tissues and improves the possibility of carrying out single cell research. The application can be used for both transcriptome research and epigenetic research.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and biotechnology, and relates to a solution for separating the nucleoplasm of adipocytes and its application. Background Technology

[0002] After more than a decade of development, single-cell sequencing technology has become an important tool in traditional biological research fields such as human diseases, species evolution, and molecular breeding. Single-cell sequencing technologies, exemplified by 10X Genomics, can now perform multi-omics detection at the single-cell level, including gene expression profiling, chromatin accessibility, and immune repertoire diversity. Furthermore, each round of experiments can simultaneously obtain genetic information from up to 10,000 cells, significantly improving the resolution of life science research.

[0003] Previously, adipose tissue was thought to perform only energy storage and some energy metabolism functions. However, with further research, it has been found that adipose tissue influences insulin sensitivity and inflammatory responses, participating in various important pathophysiological processes; adipose tissue has evolved from a simple energy storage organ into a crucial endocrine system. Therefore, single-cell research on adipose tissue is essential.

[0004] Before single-cell sequencing experiments, biological tissues need to be digested and dissociated to prepare single-cell suspensions as experimental materials. The purpose of preparing single-cell suspensions is to rapidly isolate individual cells from tissues while avoiding cell death and aggregation. The basic steps for preparing single-cell suspensions from animal tissues include: 1) breaking down solid tissue materials to increase their surface area and maximize the contact between the tissue and digestive enzymes; 2) introducing digestive enzymes to digest the extracellular matrix; and 3) cleaving the connections between cells.

[0005] Adipose tissue is composed of numerous clusters of adipocytes with little intercellular matrix. These clustered adipocytes are separated into lobules by a thin layer of loose connective tissue. Adipocytes are large in diameter, structurally fragile, and rich in lipids. Conventional single-cell suspension preparation methods cannot yield adipose tissue single-cell suspensions, and the released lipids are difficult to remove, significantly impacting the efficiency of enzymatic reactions in downstream experiments.

[0006] A literature report (Liu, X., Xiang, Q., Xu, F. et al. Single-cell RNA-seq of cultured human adipose-derived mesenchymal stem cells. Sci Data 6, 190031 (2019). https: / / doi.org / 10.1038 / sdata.2019.31) describes single-cell sequencing experiments on adipose tissue. Due to the characteristics of adipocytes, researchers chose to remove mature adipocytes during single-cell suspension preparation using methods such as centrifugation, retaining only undifferentiated adipocytes, which are mainly composed of adipose stem cells. This technique avoids the experimental challenges of adipocytes' large size, fragile structure, and high lipid content. However, this method often requires more than 50g of tissue, which is difficult to provide in experimental animals such as mice; moreover, it leads to the loss of mature adipocytes, making it impossible to conduct studies on the diversity and function of mature adipocytes. Summary of the Invention

[0007] To address the problems and / or shortcomings of existing technologies, one objective of this invention is to provide a solution capable of separating the nucleoplasm of adipocytes and its applications. This solution enables the study of all adipocyte types, increasing the possibility of conducting single-cell research.

[0008] To solve the above-mentioned technical problems, one of the technical solutions of the present invention is: a solution for separating the nucleoplasm of adipocytes, the solution comprising solution 1 and solution 2; wherein, solution 1 comprises: 8-12 mM hydrochloride buffer, 0.03-0.15% Tween-20, 8-12 mM sodium chloride, 2-4 mM magnesium chloride, 0.8-2% bovine serum albumin and 0.4-0.6% ethylphenyl polyethylene glycol;

[0009] Solution 2 comprises: 8–12 mM hydrochloride buffer, 8–25 mM sodium chloride, 1–15 mM magnesium chloride, and 0.8–2.5% bovine serum albumin; percentages are by volume.

[0010] In a preferred embodiment of the present invention, the solution 1 comprises: 10 mM hydrochloride buffer, 0.05-0.1% Tween-20, 10 mM sodium chloride, 3 mM magnesium chloride, bovine serum albumin, and 0.5% ethylphenyl polyethylene glycol; the final concentration of the bovine serum albumin is 1-2%.

[0011] In a more preferred embodiment of the present invention, the solution 1 comprises: 10 mM hydrochloride buffer, 0.1% Tween-20, 10 mM sodium chloride, 3 mM magnesium chloride, 1% bovine serum albumin and 0.5% ethylphenyl polyethylene glycol.

[0012] In a preferred embodiment of the present invention, the solution 2 comprises: 10 mM hydrochloride buffer, 10-20 mM sodium chloride, 3-10 mM magnesium chloride, and 1-2% bovine serum albumin.

[0013] In a more preferred embodiment of the present invention, the solution 2 comprises: 10 mM hydrochloride buffer, 10 mM sodium chloride, 3 mM magnesium chloride, and 1% bovine serum albumin.

[0014] To solve the above-mentioned technical problems, the second technical solution of the present invention is: a method for separating the nucleus and cytoplasm of adipocytes, the separation method comprising treating adipocytes with the solution described in the first technical solution.

[0015] In a preferred embodiment of the present invention, the separation method includes:

[0016] (1) Mix the adipose tissue with solution 1;

[0017] (2) Homogenize, freeze-incubate, and then centrifuge;

[0018] (3) After removing the supernatant, mix it with solution 2.

[0019] In a preferred embodiment of the present invention, step (1) also requires mixing with an RNase inhibitor; the RNase inhibitor includes diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex, or a protein inhibitor of RNase.

[0020] In a preferred embodiment of the present invention, the homogenization is performed by pressing and twisting the grinding rod in step (2), and the homogenization time is preferably 2 to 3 minutes, more preferably 2 minutes.

[0021] In a preferred embodiment of the present invention, the freezing incubation in step (2) is a freezing incubation at -20 to -25°C for 5 to 10 minutes, preferably a freezing incubation at -22°C for 8 minutes.

[0022] In a preferred embodiment of the present invention, the centrifugation in step (2) is 300-1000g for 1-5 minutes, preferably 500g for 2 minutes or 1000g for 1 minute.

[0023] In a more preferred embodiment of the present invention, the separation method further includes purification; the purification is flow cytometry purification; the flow cytometry purification includes filtration, staining and sorting.

[0024] In a more preferred embodiment of the present invention, the filtration includes filtering the crude cell nucleus suspension obtained in the separation method described in technical solution two using a 40 μm filter screen.

[0025] In a more preferred embodiment of the present invention, the staining includes adding 5 μl of nucleic acid dye to the filtered crude cell nucleus suspension, incubating at room temperature for 8–12 min, and centrifuging and resuspending; the nucleic acid dye is propidium iodide, phenylindole, or 7-aminoactinomycin D.

[0026] In a preferred embodiment of the present invention, the mixture is centrifuged and resuspended to 500 μl to obtain a centrifuged resuspension.

[0027] In a preferred embodiment of the present invention, the sorting includes loading the centrifuged resuspension onto a flow cytometer, sorting an appropriate amount of cell nuclei, and collecting them into 500 μl to 1000 μl of solution 2 as defined in one of the technical solutions.

[0028] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0029] The reagents and raw materials used in this invention are all commercially available.

[0030] This invention discloses an experimental method for single-cell sequencing of adipose tissue. The experimental procedure includes: 1) dissociating the tissue, removing lipids, and releasing the adipocyte nuclei; 2) purifying the high-quality single-cell nucleus suspension by flow cytometry; and 3) conducting single-cell sequencing experiments.

[0031] The positive and progressive effects of this invention are as follows:

[0032] 1. Compared with existing technologies that cannot study mature adipocytes, this invention can enable the study of all cell types in adipose tissue.

[0033] 2. This invention significantly reduces the amount of adipose tissue required (less than 200 mg), far less than other existing methods, and does not lose mature adipocytes, thus increasing the possibility of conducting single-cell studies (such as surgically sampled periprostatic adipose tissue, puncture-obtained perithyroid adipose tissue, etc.).

[0034] 3. Compared with existing technologies that only focus on one aspect, this invention can be used for both transcriptome research and epigenetics research. Attached Figure Description

[0035] Figure 1 A gating protocol for flow cytometry to sort single-cell nucleus suspensions.

[0036] Figure 2 Differences in the detection of different nucleic acid dyes on flow cytometry.

[0037] Figure 3 Microscopic examination results of single-cell nucleus suspensions of backfat adipose tissue from domestic pigs.

[0038] Figure 4The results of 2100 detection of cDNA products in single-cell nuclear RNA sequencing of backfat adipose tissue from domestic pigs.

[0039] Figure 5 The results of microscopic examination of a single-cell nucleus suspension from the abdominal adipose tissue of mice.

[0040] Figure 6 The results of PCR product detection in single-cell ATAC sequencing of mouse abdominal adipose tissue are shown in Figure 2100. Detailed Implementation

[0041] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0042] Example 1: Dissociation of tissue, removal of lipids, and release of adipocyte nuclei

[0043] (1) Prepare solution 1: 10 mM hydrochloride buffer (INVITROGEN, 15567-027), 0.1% Tween-20 (BBI, T0777-500ML), 10 mM sodium chloride (SIGMA, 71376), 3 mM magnesium chloride (SIGMA, M2393); then add 1% bovine serum albumin (SIGMA, B4287) and 0.5% ethyl phenyl polyethylene glycol (SIGMA, 74385-500ML) to the final concentration; prepare solution 2: 10 mM hydrochloride buffer, 10 mM sodium chloride, 3 mM magnesium chloride, 1% bovine serum albumin.

[0044] (2) Take a block of adipose tissue, place it on multiple layers of paper towels, and squeeze to remove some of the fat from the tissue;

[0045] (3) Weigh no more than 200 mg of fresh / frozen tissue and cut it into small pieces (approximately 2 × 2 mm). Open the Minute Adipose Tissue Cytoplasmic-Nucleus Separation Kit (Invent Biotechnologies, AN-029), transfer the tissue to the bottom of a 1.5 ml tube, and add 600 μl of Solution 1; if performing RNA sequencing experiments, add an additional 3 μl of RNase inhibitor (20 U / μl, Thermo Fisher Scientific, AM2694);

[0046] (4) Press and twist the grinding rod to homogenize the tissue for 2-3 minutes;

[0047] (5) Insert the centrifuge column into the receiving tube and pour the homogenized liquid into the centrifuge column sleeve;

[0048] (6) Open the centrifuge column and incubate at -22°C for 8 minutes. After incubation, centrifuge at 500g for 2 minutes; if there is liquid residue in the centrifuge column, increase the centrifugal force to 1000g and centrifuge for 1 minute.

[0049] (7) Discard the centrifuge column, cap it, and vortex to mix. Centrifuge at 1000g for 4 minutes;

[0050] (8) Discard the supernatant, add 50 μl of solution 2, and mix by pipetting; if performing RNA sequencing experiments, add an additional 3 μl of RNase inhibitor.

[0051] Table 1 shows the gradient experiments for solution 1, with other reagents as shown in Example 1. Table 2 shows the gradient experiments for solution 2. The experimental results show that solution 2 in the buffer formulation of this invention can maintain the cell nucleus structure for more than 15 minutes. As can be seen from Table 1, the concentration ratio of solution 1 can maximize the number of intact cell nuclei obtainable per unit of adipose tissue. As can be seen from Table 2, the concentration of solution 2 can maintain the morphological structure and nucleic acid stability of the cell nucleus. A longer maintenance time is more beneficial for downstream experiments; if the experimental materials are not under optimal storage conditions, it may significantly interfere with the results of downstream experiments.

[0052] Table 1. Component Gradient Experiment of Solution 1

[0053]

[0054] Table 2. Gradient Experiment of Two Components in Solution

[0055]

[0056] Example 2: Flow cytometry purification yielded a high-quality single-cell nucleus suspension.

[0057] (1) The crude cell nucleus suspension obtained in Example 1 was filtered through a 40 μm filter and observed under a microscope;

[0058] (2) After filtering the crude cell nucleus suspension, add nucleic acid dye to label it, add 5 μl of propidium iodide (INVITROGEN, BMS500PI), or 5 μl of phenylindole, or 5 μl of 7-aminoactinomycin D staining solution, incubate at room temperature for 10 min, centrifuge and resuspend to 500 μl.

[0059] (3) Using a flow cytometer (SONY, SH800S), sort an appropriate number of cell nuclei and collect them into 500 μl of solution 2. If performing RNA sequencing experiments, add an additional 3 μl of RNase inhibitor; see the gating protocol. Figure 1 , Figure 1The flow cytometer gating scheme for single-cell nucleus suspension sorting in this invention is shown in the left figure, which shows the delineation of single-cell nucleus groups by scatter plot of forward and side light, the middle figure shows the delineation of non-adhesive single-cell nucleus groups by area-height plot of forward light, and the right figure shows the delineation of single-cell nucleus groups to be sorted by positive signal of propidium iodide staining.

[0060] Figure 2 The differences in detection by different nucleic acid dyes on flow cytometry are shown. The left image shows propidium iodide, the middle image shows phenylindole, and the right image shows 7-aminoactinomycin D. Phenylindole staining results showed weak positive peaks and poor specificity, leading to a large amount of cell debris in the sorting results. 7-aminoactinomycin D showed weak fluorescence signals, with a positive signal ratio of only 61.3% in the PI group, resulting in low nuclear recovery efficiency.

[0061] Example 3: Preparation of single-cell nuclear suspension from backfat adipose tissue of domestic pigs for single-cell RNA sequencing experiments.

[0062] (1) Take about 200mg of back fat tissue from domestic pigs and prepare a single-cell nucleus suspension using the technical solutions of Examples 1 and 2 of this invention;

[0063] (2) Take 5 μl of the prepared single-cell nucleus suspension, add 5 μl of trypan blue (Beyond, C0011) staining solution, and observe under a microscope. Figure 3 The image shows the microscopic examination results of a single-cell nucleus suspension from the backfat adipose tissue of a domestic pig. In the image, A represents a complete nuclear structure, while B and C represent cell fragments.

[0064] (3) The cell counting chamber yielded 120,000 single cell nuclei, with a fragmentation rate of less than 10%. Figure 3 The quality is good and it can be used for downstream sequencing experiments.

[0065] (4) Take 16,000 single-cell nuclei to conduct single-cell RNA sequencing experiments based on 10X Genomics.

[0066] A prepared single-nucleus suspension was used to encapsulate gel beads with cell-tagged sequences and cell nuclei within droplets using a microfluidic chip. Within the droplets, the cell nuclei ruptured, releasing mRNA that linked to the cell-tagged sequences on the gel beads, forming single-nucleus GEMs. The nuclear mRNA underwent reverse transcription within the droplets to form cDNA, followed by demulsification for library construction. After the library passed quality control, sequencing was performed using an MGISEQ-2000 sequencer.

[0067] The cDNA product was analyzed using an Agilent 2100 bioanalyzer, and the results are shown below. Figure 4 , Figure 4 The results show that when the main peak is greater than 1000bp, the experimental results are considered reliable, indicating that there is no significant degradation of RNA in the cell nucleus. The main peak in the figure is 1082bp.

[0068] The results show that, using this invention, a suspension of cell nuclei from all cells in adipose tissue can be obtained efficiently. The cell nuclei have intact structures and the nuclear RNA has not been significantly degraded, allowing for the full extraction of gene expression profile information they carry.

[0069] (5) The library was sequenced using the DNBSEQ platform, yielding 10,758 single-cell data points. After dimensionality reduction analysis of the data matrix, information on 14 cell subpopulations was obtained. The results indicate that using this invention, the nuclei of all cells in adipose tissue can be obtained efficiently without losing information such as mature adipocytes, thus improving data utilization.

[0070] Example 4: Preparation of single-cell nucleus suspension from mouse abdominal adipose tissue for single-cell ATAC sequencing experiments.

[0071] (1) Take about 200 mg of abdominal adipose tissue from a mouse and prepare a single-cell nucleus suspension using the technical solutions of Examples 1 and 2 of this invention;

[0072] (2) Take 5 μl of the prepared single-cell nucleus suspension, add 5 μl of trypan blue (Beyond, C0011) staining solution, and observe under a microscope. Figure 5 This image shows the microscopic examination results of a single-cell nucleus suspension from mouse abdominal adipose tissue. In the image, A represents a complete nuclear structure, while B and C represent cell debris; the results are as follows. Figure 5 As shown, 150,000 single-cell nuclei were obtained, with a fragmentation rate of less than 10%; the quality is good and can be used for downstream sequencing experiments.

[0073] (3) The experimental group obtained 150,000 single cell nuclei with a fragmentation rate of less than 10%; the quality was good and could be used for downstream sequencing experiments.

[0074] (4) Take 16,000 single cell nuclei to carry out single-cell ATAC sequencing experiments based on 10X Genomics.

[0075] The prepared single-cell nucleus suspension was added to the Tn5 enzyme reaction system. The Tn5 enzyme entered the nucleus and cleaved open chromatin regions. Using a microfluidic chip, gel beads with cell-tagged sequences and cell nuclei were encapsulated in droplets. In the droplets, the released chromatin fragments linked to the cell-tagged sequences on the gel beads, forming single-cell GEMs structures. Subsequently, emulsion was broken, and libraries were constructed from the chromatin fragments with adapters. After the libraries passed quality control, sequencing was performed using an MGISEQ-2000 sequencer.

[0076] The amplification products were analyzed using an Agilent 2100 bioanalyzer, and the results are shown below. Figure 6 , Figure 6The results show that when there are more than three main peaks, the experimental results are considered normal, and there is no obvious damage to the chromatin structure in the cell nucleus. The figure contains three peaks, including one at 351 bp.

[0077] The results show that, using this invention, a suspension of cell nuclei from all cells in adipose tissue can be obtained efficiently. The cell nuclei have intact structures and the spatial structure of the chromatin within the nuclei is not significantly damaged, allowing for the full extraction of the epigenetic information they carry.

[0078] (5) The library was sequenced using the DNBSEQ platform, yielding 7497 single-cell data points. Dimensionality reduction analysis of the data matrix revealed 16 cell subpopulations. The results indicate that this invention can efficiently obtain nuclear suspensions of all cells within adipose tissue without losing information such as mature adipocytes, thus improving data utilization.

Claims

1. The application of a solution in the separation of adipocyte nucleoplasm, characterized in that, The solution comprises solution 1 and solution 2; wherein, Solution 1 comprises: 10 mM hydrochloride buffer, 0.05-0.1% Tween-20, 10 mM sodium chloride, 3 mM magnesium chloride, 1-2% bovine serum albumin and 0.5% ethylphenyl polyethylene glycol; Solution 2 comprises: 10 mM hydrochloride buffer, 10-20 mM sodium chloride, 3-10 mM magnesium chloride, and 1-2% bovine serum albumin; percentages are by volume.

2. The application as described in claim 1, characterized in that, Solution 1 comprises: 10 mM hydrochloride buffer, 0.1% Tween-20, 10 mM sodium chloride, 3 mM magnesium chloride, 1% bovine serum albumin and 0.5% ethylphenyl polyethylene glycol; Solution 2 contains: 10 mM hydrochloride buffer, 10 mM sodium chloride, 3 mM magnesium chloride and 1% bovine serum albumin.

3. A method for isolating adipocyte nucleoplasm, the method comprising treating adipocytes with a solution as described in claim 1 or 2.

4. The separation method as described in claim 3, characterized in that, The separation method includes: (1) Mix the adipose tissue with solution 1; (2) Homogenize, freeze-incubate, and then centrifuge; (3) After removing the supernatant, mix it with solution 2.

5. The separation method as described in claim 4, characterized in that, In step (1), it is also necessary to mix with an RNase inhibitor; the RNase inhibitor includes diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex or protein inhibitors of RNase. And / or, in step (2), the homogenization is performed by pressing and twisting with a grinding rod; And / or, the freezing incubation in step (2) is a freezing incubation at -20~-25℃ for 5~10 minutes; And / or, the centrifugation in step (2) is 300~1000g, centrifugation for 1~5min.

6. The separation method as described in claim 5, characterized in that, In step (2), the homogenization time by pressing and twisting the grinding rod is 2 to 3 minutes; and / or, the freezing incubation in step (2) is -22℃ freezing incubation for 8 minutes; and / or, the centrifugation in step (2) is 500g centrifugation for 2 minutes or 1000g centrifugation for 1 minute.

7. The separation method as described in claim 5 or 6, characterized in that, In step (2), the time for homogenizing by pressing and twisting the grinding rod is 2 minutes.

8. The separation method as described in claim 3, characterized in that, The separation method further includes purification; the purification is flow cytometry purification; the flow cytometry purification includes filtration, staining and sorting.

9. The separation method as described in claim 8, characterized in that, The filtration includes filtering the crude cell nucleus suspension obtained in the separation method of claim 3 using a 40 μm filter screen.

10. The separation method as described in claim 8, characterized in that, The staining process involves adding 5 μl of nucleic acid dye to the filtered crude cell nucleus suspension, incubating at room temperature for 8–12 min, and then centrifuging and resuspending. The nucleic acid dye is propidium iodide, phenylindole, or 7-aminoactinomycin D.

11. The separation method as described in claim 10, characterized in that, Centrifuge and resuspend to 500 μl to obtain a centrifuged resuspended solution.

12. The separation method as described in claim 11, characterized in that, The sorting process includes loading the centrifuged resuspension onto a flow cytometer, sorting an appropriate number of cell nuclei, and collecting them into 500 μl to 1000 μl of solution 2 as defined in claim 1.

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