Method for separating, culturing and purifying olfactory bulb and olfactory ensheathing cells

Abstract

The invention relates to the technical field of animal cell culture, in particular to a method for separating, culturing and purifying olfactory bulb and olfactory ensheathing cells. The method specifically relates to the steps of isolated culture and purification, and a differential adhesion method, a cytarabine purification method and a serum-free culture purification method are combined in the purification step, so that the olfactory ensheathing cells with high purity and high cell viability are obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of animal cell culture, in particular to a method for separating, culturing and purifying olfactory bulb and olfactory ensheathing cells. Background Art

[0002] In the field of neuroscience research, olfactory ensheathing cells (OECs) in the olfactory bulb have shown great potential in the repair of nervous system injuries due to their unique ability to promote neuroregeneration. OECs are a special type of glial cell that are primarily found in the olfactory mucosa and olfactory bulb. Obtaining single OECs primarily relies on isolating them from the olfactory mucosa or olfactory bulb, making their isolation, culture, and purification techniques crucial.

[0003] Chinese Patent Publication No. CN111019895A, entitled "A Method for Purifying and Cultivating Olfactory Ensheathing Cells," discloses a method for purifying and culturing OECs. By using complete culture medium containing forskolin and repeated digestion, OECs with a purity exceeding 98% were obtained. However, OECs are sensitive to enzymatic digestion and are prone to severe damage during enzymatic digestion, resulting in impaired growth and proliferation. Wang Bin et al. (In vitro Culture of Human Embryonic Olfactory Ensheathing Cells Using Different Purification Methods, Journal of Sichuan University (Medical Edition), 2008) also noted that the use of cytarabine to purify OECs resulted in massive cell death and shedding, ultimately leading to purification and culture failure. Furthermore, serum-free culture methods failed to improve OEC purity and resulted in a decline in cell status. The technical challenges of isolating, culturing, and purifying OECs are high, and the low purity and activity of the purified cells have long been a major challenge in this field. Summary of the Invention

[0004] In order to facilitate those skilled in the art to better understand the technical solutions of the present invention, the present invention provides explanations for the following abbreviations and terms.

[0005] FBS: Fetal bovine serum, which provides rich growth factors, hormones, carrier proteins, etc., and provides necessary nutrients and signaling molecules for cell growth, proliferation and survival.

[0006] DF12: A culture medium that provides cells with basic nutrients, including amino acids, vitamins, inorganic salts, etc., to maintain normal cell metabolism and physiological functions.

[0007] Hanks solution: A balanced salt solution that does not contain calcium and magnesium ions, used for rinsing tissue blocks and cells when collecting samples for cell culture.

[0008] DMEM: high glucose medium.

[0009] PLL: Poly-L-lysine, poly L-lysine, has a positive charge and can bind to the surface of the culture flask, making the surface of the culture flask positively charged, thereby promoting negatively charged cells to better adhere to the surface of the culture flask, which is beneficial to the cell's adherent growth.

[0010] The purpose of the present invention is to provide a method for isolating, culturing and purifying olfactory ensheathing cells of the olfactory bulb, which has the advantages of being simple, economical and fast, and can effectively remove and inhibit the growth of fibroblasts, thereby obtaining high-purity olfactory ensheathing cells.

[0011] In view of this, the present invention provides a method for isolating, culturing and purifying olfactory ensheathing cells of the olfactory bulb, comprising isolation, culture and purification, wherein the isolation and culture comprises the steps of removing the olfactory nerve layer and the glomerular layer on the surface of the olfactory bulb, and obtaining a cell suspension containing olfactory ensheathing cells after trypsin digestion; the purification comprises a differential adherence culture stage, a cytarabine inhibition culture stage and a purification liquid inhibition culture stage, wherein after the fibroblasts and astrocytes are removed from the cell suspension in the differential adherence culture stage, the cell suspension is inoculated in an inhibition culture medium containing cytarabine for inhibition culture, and then the inhibition culture medium is replaced with a serum-free purification liquid culture medium to continue the inhibition culture to obtain purified olfactory ensheathing cells; wherein the concentration of cytarabine in the inhibition culture medium is 0.4-0.8 μg / mL.

[0012] Olfactory ensheathing cells are mainly concentrated in the olfactory nerve layer and the glomerular layer on the surface of the olfactory bulb. Separating the olfactory nerve layer and the glomerular layer from the surface of the olfactory bulb first is beneficial to preparing a cell suspension with a high content of olfactory ensheathing cells. The method of preparing tissue into a cell suspension is a conventional method in the field. For example, Wang Bin et al. provided a method for making the olfactory nerve layer and the glomerular layer into a single cell suspension: the olfactory nerve layer and the glomerular layer were cut into pieces of about 0.5mm×0.5mm in size, and digested with 0.25% trypsin at 37°C for 15 minutes. The digestion was terminated with a culture medium containing 10% FBS for 5 minutes, centrifuged at 100rpm for 5 minutes, and the supernatant was removed. DF12 culture medium was added, gently blown 15 to 20 times and filtered through a 200-mesh filter to prepare a single-cell suspension. The present invention also provides a method for making the olfactory nerve layer and the glomerular layer into a single-cell suspension: the olfactory nerve layer and the glomerular layer were rinsed twice with D-Hanks solution, cut into pieces of 0.5 to 1mm 2The cells were digested with 0.25% trypsin at 37°C for 15 minutes, and gently pipetted to form a cell suspension. DMEM containing 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin was added to terminate the reaction. The supernatant was removed by centrifugation, and the cells were washed twice with D-Hanks solution and resuspended in 1 mL of complete culture medium to obtain a single-cell suspension of olfactory ensheathing cells. It should be understood by those skilled in the art that the method for preparing a cell suspension provided by the present invention does not constitute a limitation to the technical solution of the present invention, but is only intended to provide a variety of different methods for preparing a cell suspension to fully support the source of the cell suspension in the claims of the present invention.

[0013] Differential adhesion is a commonly used technique for purifying cells in the art. Nash has published the technical principles and key operational points of the differential adhesion method (Nash HH, Borke RC, Anders JJ. New method of purification for establishing primary cltures of ensheathing cells from the adult olfactory bulb. GLIA, 2001; 34(2): 81-87). Wang Bin et al. also pointed out that when culturing OECs, fibroblasts and astrocytes can be separated from OECs by utilizing the differences in their attachment time. The present invention also provides a method for purifying OECs using the differential adhesion method: - Plate the cell suspension onto uncoated PLL-containing flasks containing DEME and incubate at 37°C, 5% CO2 for 12 hours; this process will remove most of the fibroblasts.

[0014] - Take the supernatant and inoculate it onto a culture flask containing DEME without PLL coating, and continue to culture at 37°C, 5% CO2 for 12 hours; this process can remove most of the astrocytes, and OECs will be present in the supernatant.

[0015] The present invention combines the cytarabine purification method with the serum-free starvation method to obtain OECs with strong activity and high purity, which not only overcomes the defects of OECs' intolerance to cytarabine, but also overcomes the defects of low activity and low purity of OECs during serum-free culture.

[0016] In some embodiments, the concentration of cytarabine in the inhibition medium is 0.6 μg / mL.

[0017] Cytarabine is toxic to OECs, so controlling the concentration of cytarabine is crucial. After extensive creative work, the inventors discovered that OECs are minimally affected by cytarabine at a concentration of 0.6 μg / mL and can maintain the highest cell activity.

[0018] In some embodiments, the inhibitory medium comprises FBS, DF12 medium, and cytarabine.

[0019] In some embodiments, the volume ratio of FBS in the inhibition medium is 10%, and the working concentration of cytarabine in the inhibition medium is 0.6 μg / mL.

[0020] In some embodiments, the purified liquid culture medium comprises DMEM, N2 cell culture supplement, biotin, L-glutamine, penicillin, and streptomycin.

[0021] In the present invention, the term "N2 cell culture supplement" is a formulated reagent specifically for neural cell culture, and is commonly used in serum-free culture of neural cells.

[0022] In some embodiments, the N2 cell culture supplement consists of transferrin, sodium selenate, insulin, progesterone, and putrescine.

[0023] In some embodiments, during the cytarabine inhibition culture phase, once the olfactory ensheathing cells are observed to have completed adherent growth, the purified medium is used for an 80% medium exchange, and the purified medium inhibition culture phase is entered. In some embodiments, the cytarabine inhibition culture phase does not exceed 48 hours. By strictly controlling the duration of the cytarabine inhibition culture phase, the toxic effects of cytarabine on OECs can be effectively reduced, thereby achieving the effect of increasing OEC activity.

[0024] In some embodiments, during the purified solution inhibition culture stage, the purified solution culture medium is completely replaced every 3 days.

[0025] In some embodiments, the separation culture, the differential adherence culture, the cytarabine inhibition culture and the purified solution inhibition culture are all carried out at 37° C. and 5% CO 2 .

[0026] As used herein, the term "stop solution" refers to a reagent that can be used to terminate trypsin digestion, typically containing serum proteins that bind to and inactivate trypsin. Therefore, the stop solution can be a saline buffer solution containing only serum proteins, a saline buffer solution containing serum components, or any other solution or reagent that is non-toxic to cells and can inactivate trypsin.

[0027] The present invention has the following advantages and effects: The separation, culture and purification method of the present invention can obtain highly active and highly pure OECs, with a purity as high as 95% within 7 days of culture.

[0028] The present invention overcomes the defect that cytarabine cannot be used to purify OECs, and successfully fills the technical gap of purifying OECs by cytarabine.

[0029] The method is simple and quick to operate, uses low-priced reagents, and has high economic and practical value. DETAILED DESCRIPTION

[0030] To make the objects, features, and advantages of the present invention more readily apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental reagents and materials used in the present invention can be obtained commercially.

[0032] Examples 1-3 Olfactory ensheathing cells are obtained by the following isolation, culture and purification methods: S1. Remove the olfactory bulb tissue. Quickly separate the olfactory tract from the olfactory bulb under sterile conditions and place it in pre-chilled calcium- and magnesium-free Hanks' balanced salt solution. Under a dissecting microscope, use fine forceps and a blade to carefully peel away the pia mater and vascular tissue from the surface of the olfactory bulb. Then, separate the olfactory nerve layer and the glomerulus layer.

[0033] S2. Rinse the separated tissue twice with Hanks solution and cut into pieces of about 0.5 mm 2 Keep the tissue on ice throughout the entire process.

[0034] S3. Add the tissue blocks to 0.25% trypsin digestion buffer and digest in a 37°C, 5% CO2 incubator for 15 minutes. Gently pipette every 5 minutes to fully disaggregate the tissue blocks into a cell suspension. After digestion, add an appropriate amount of stop solution to terminate the reaction. Transfer the cell suspension to a centrifuge tube and centrifuge at 1200 rpm for 5 minutes at room temperature. Discard the supernatant and wash the cell pellet twice with Hanks' solution. After centrifugation, add 1 mL of complete culture medium to resuspend the cells. Mix one drop of cell suspension with one drop of trypan blue stain and add the resulting mixture to a hemocytometer for counting.

[0035] S4. Inoculate the above cell suspension onto a culture flask not coated with PLL but containing DEME, and culture in a 37°C, 5% CO2 incubator for 12 h.

[0036] S5. After the incubation period, gently aspirate the supernatant and transfer it to a new culture flask containing DEME, not coated with PLL, and continue incubation for 12 hours. During this time, prepare the inhibitory medium and the purified medium, sterilize them, and set aside. The recipes for the inhibitory medium and purified medium are shown in Table 1.

[0037] S6. After the culture is completed, the supernatant is inoculated into the inhibition medium and then inoculated into the culture flask coated with PLL.

[0038] S7. Within 48 hours after inoculation, the olfactory ensheathing cells were observed to have completed their adherent growth. At this time, 80% of the culture medium was replaced with purified medium to remove non-adherent impurity cells and residual cytarabine.

[0039] S8. Place the culture flask at 37°C and 5% CO2 to obtain purified OECs. Purified OECs can be cultured continuously, but the culture medium must be completely replaced with purified medium every three days.

[0040] Table 1 Culture medium formula The formula of N2 Cell Culture Supplement is as follows: Insulin 500 μg / mL; transferrin 500 μg / mL; sodium selenate 0.52 μg / mL; progesterone 0.63 μg / mL; putrescine 1611 μg / mL.

[0041] Comparative Example 1-2 Purification of Cytarabine Olfactory ensheathing cells are obtained by the following isolation, culture and purification methods: S1. Remove the olfactory bulb tissue. Quickly separate the olfactory tract from the olfactory bulb under sterile conditions and place it in pre-chilled calcium- and magnesium-free Hanks' balanced salt solution. Under a dissecting microscope, use fine forceps and a blade to carefully peel away the pia mater and vascular tissue from the surface of the olfactory bulb. Then, separate the olfactory nerve layer and the glomerulus layer.

[0042] S2. Rinse the separated tissue twice with Hanks solution and cut into pieces of about 0.5 mm 2 Keep the tissue on ice throughout the entire process.

[0043] S3. Add the tissue blocks to 0.25% trypsin digestion buffer and digest in a 37°C, 5% CO2 incubator for 15 minutes. Gently pipette every 5 minutes to fully disaggregate the tissue blocks into a cell suspension. After digestion, add an appropriate amount of stop solution to terminate the reaction. Transfer the cell suspension to a centrifuge tube and centrifuge at 1200 rpm for 5 minutes at room temperature. Discard the supernatant and wash the cell pellet twice with Hanks' solution. After centrifugation, add 1 mL of complete culture medium to resuspend the cells. Mix one drop of cell suspension with one drop of trypan blue stain and add the resulting mixture to a hemocytometer for counting.

[0044] S4. Inoculate the above cell suspension onto a culture flask not coated with PLL but containing DEME, and culture in a 37°C, 5% CO2 incubator for 12 h.

[0045] S5. After the culture is completed, gently aspirate the supernatant and transfer it to a new culture flask not coated with PLL and containing DEME, and continue to culture for 12 hours.

[0046] S6. After the culture, the supernatant was inoculated into an inhibition medium with a final cytarabine concentration of 2 μg / mL (Comparative Example 1) and 0.6 μg / mL (Comparative Example 2), and then inoculated into a culture flask coated with PLL and cultured for 48 h.

[0047] S7. After the culture is completed, the supernatant is discarded, the cell pellet is washed twice with Hanks solution, and the cell pellet is added to DF12 medium containing 10% FBS, 20 μmol / L Forskolin, and 20 μg / L BPE (bovine pituitary extract) to obtain purified olfactory ensheathing cells.

[0048] Comparative Example 3: Purification by serum-free starvation method Olfactory ensheathing cells are obtained by the following isolation, culture and purification methods: S1. Remove the olfactory bulb tissue. Quickly separate the olfactory tract from the olfactory bulb under sterile conditions and place it in pre-chilled calcium- and magnesium-free Hanks' balanced salt solution. Under a dissecting microscope, use fine forceps and a blade to carefully peel away the pia mater and vascular tissue from the surface of the olfactory bulb. Then, separate the olfactory nerve layer and the glomerulus layer.

[0049] S2. Rinse the separated tissue twice with Hanks solution and cut into pieces of about 0.5 mm 2 Keep the tissue on ice throughout the entire process.

[0050] S3. Add the tissue blocks to 0.25% trypsin digestion buffer and digest in a 37°C, 5% CO2 incubator for 15 minutes. Gently pipette every 5 minutes to fully disaggregate the tissue blocks into a cell suspension. After digestion, add an appropriate amount of stop solution to terminate the reaction. Transfer the cell suspension to a centrifuge tube and centrifuge at 1200 rpm for 5 minutes at room temperature. Discard the supernatant and wash the cell pellet twice with Hanks' solution. After centrifugation, add 1 mL of complete culture medium to resuspend the cells. Mix one drop of cell suspension with one drop of trypan blue stain and add the resulting mixture to a hemocytometer for counting.

[0051] S4. The cell suspension was seeded onto a culture flask containing DEME, not coated with PLL, and cultured in a 37°C, 5% CO2 incubator. When rapid fibroblast growth was observed under a microscope, the culture medium was changed (using the purified culture medium described in Example 1). After the fibroblasts detached, the culture medium was changed (using DF12 culture medium containing 10% FBS) to obtain purified olfactory ensheathing cells.

[0052] Test case immunohistochemistry identification experiment 1.1 Reagents and Materials 4% paraformaldehyde (freshly prepared), PBS, blocking buffer, rabbit anti-human P75NGFR polyclonal antibody, mouse anti-human S-100 monoclonal antibody, goat anti-rabbit 546, goat anti-mouse 488, Hoechst.

[0053] 1.2 Cell samples to be tested The olfactory ensheathing cells of the embodiment, the olfactory ensheathing cells of comparative example 1 and the olfactory ensheathing cells of comparative example 2.

[0054] 1.3 Experimental Methods Prepare a primary antibody mixture consisting of rabbit anti-human P75NGFR polyclonal antibody (1:200) and mouse anti-human S-100 monoclonal antibody (1:200) for later use; Prepare a secondary antibody mixture consisting of goat anti-rabbit 546 (1:100) and goat anti-mouse 488 (1:100) and set aside; Cell specimens cultured for 7 and 14 days were fixed with 4% paraformaldehyde for 20 minutes and then rinsed three times with PBS for 5 minutes each. After blocking with goat serum (blocking solution) at room temperature for 30 minutes, the primary antibody mixture was added and the cells were incubated at 37°C for 1 hour. The cells were then rinsed three times with PBS for 5 minutes each. The secondary antibody mixture was then added and the cells were incubated at 37°C for 30 minutes. The cells were then rinsed five times with PBS for 5 minutes each. Hoechst was then added and incubated at 37°C for 2 minutes. The cells were then rinsed three times with PBS for 5 minutes each. The cell specimens were then placed under a 200x fluorescence microscope under the corresponding excitation light. The staining results for P75NGFR, S-100, and Hoechst were observed, and the positive cell rate was calculated using the following formula: Positive cell rate = (number of cells co-stained with P75NGFR and S-100 / number of cells stained with Hoechst)*100%.

[0055] 1.4 Experimental Results Both P75NGFR and S-100 are cytokines specifically expressed by OECs. Therefore, cells showing both P75NGFR and S-100 fluorescence are considered OECs. Hoechst is a nuclear stain that stains the nuclei of all cells and produces a corresponding fluorescence reaction. Therefore, within the same microscopic field, the ratio of cells showing both P75NGFR and S-100 fluorescence to the number of cells stained with Hoechst is the OECs positivity rate. The results are shown in Table 2.

[0056] Table 2 Positive cell rate The results showed that after 7 days of culture, the positive rates of the OECs from Examples 1-3 all reached 95%, while all the OECs from Comparative Example 1 had died. The positive rate of the OECs from Comparative Example 2 was 20.7%, with numerous fibroblasts visible, demonstrating that the purification of OECs from Comparative Example 2 was poor and nearly ineffective. The positive rate of the OECs from Comparative Example 3 was 82.8%. After 14 days, the positive rates of the OECs from Examples 1-3 remained above 80%, while the positive rate of the OECs from Comparative Example 3 had dropped to 22.1%.

[0057] These results demonstrate that OECs obtained using the isolation, culture, and purification methods of the present invention possess higher purity, reaching 95% purity within 7 days. Furthermore, the OECs obtained using the isolation, culture, and purification methods of the present invention exhibit enhanced cell viability, retaining 80% purity after 14 days of culture. These results demonstrate that the isolation, culture, and purification methods of the present invention can yield OECs of high purity and viability.

[0058] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they become aware of the basic creative concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.

[0059] Finally, it should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or terminal device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of additional identical elements in the process, method, article, or terminal device that includes the element.

[0060] The present invention has been introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core ideas. At the same time, for those skilled in the art, according to the ideas of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A method for isolating, culturing and purifying olfactory bulb and olfactory ensheathing cells, comprising isolation, culturing and purification, characterized in that: The isolation and culture comprises the steps of removing the olfactory nerve layer and the glomerular layer on the surface of the olfactory bulb, and digesting them with trypsin to obtain a cell suspension containing olfactory ensheathing cells; The purification includes a differential adherence culture stage, a cytarabine inhibition culture stage, and a purification solution inhibition culture stage. After the cell suspension undergoes the differential adherence culture stage to remove fibroblasts and astrocytes, it is inoculated in an inhibition culture medium containing cytarabine for inhibition culture. The inhibition culture medium is then replaced with a serum-free purification solution culture medium to continue the inhibition culture, thereby obtaining purified olfactory ensheathing cells. Wherein, the concentration of cytarabine in the inhibition culture medium is 0.4-0.8 μg / mL.

2. The method according to claim 1, characterized in that The concentration of cytarabine in the inhibition medium was 0.6 μg / mL.

3. The method according to claim 1, characterized in that The inhibitory culture medium includes FBS, DF12 culture medium and cytarabine.

4. The method according to claim 3, characterized in that The volume ratio of FBS in the inhibition medium is 10%, and the working concentration of cytarabine in the inhibition medium is 0.6 μg / mL.

5. The method according to claim 1, wherein The purified liquid culture medium comprises DMEM, N2 cell culture additive, biotin, L-glutamine, penicillin and streptomycin.

6. The method according to claim 5, characterized in that The N2 cell culture additive consists of transferrin, sodium selenate, insulin, progesterone and putrescine.

7. The method according to claim 1, characterized in that In the cytarabine inhibition culture stage, after the olfactory ensheathing cells were observed to have completed their adherent growth, the purified medium was used to perform an 80% medium change, and the purified medium inhibition culture stage was entered.

8. The method according to claim 7, characterized in that The cytarabine inhibition culture period does not exceed 48 hours.

9. The method according to claim 7, characterized in that During the purified solution inhibition culture stage, the purified solution culture medium was completely replaced every 3 days.

10. The method according to any one of claims 1 to 9, characterized in that The separation culture, the differential adherence culture, the cytarabine inhibition culture and the purified liquid inhibition culture were all carried out at 37° C. and 5% CO 2 .

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