PREPARATION METHOD FOR OLFACTORY PRECURSOR CELL

Abstract

Provided is a preparation method for olfactory progenitor cells. Also provided is an olfactory progenitor cell obtained by the method according to the present invention, wherein the single cell of the olfactory progenitor cell can be serially passaged for more than 11 generations. Compared with the prior art, the preparation method for olfactory progenitor cells of the present invention has excellent effects, by which a large quantity of olfactory progenitor cells can be obtained. Moreover, the method is simple and feasible with low cost and good safety, and has a good application prospect in China and abroad.

Claims

1. A preparation method for olfactory progenitor cells, comprising the following steps of: (1) taking the olfactory mucosa tissues between the upper turbinate and middle turbinate, and subjecting them to an aseptic treatment; (2) pretreating the olfactory mucosa tissue blocks obtained in step (1) with 1-2 times volume of 5% (g/100 mL) trypsin aqueous solution and 1-2 times volume of 0.2 mmol/L EDTA aqueous solution at 36-38° C. for 5-15 minutes; (3) removing the loose layers of surface and base membranes of the olfactory mucosa tissue blocks pretreated in step (2), rinsing the olfactory mucosa tissue blocks with PBS for 2-5 times, and preparing them into small pieces with a size of 0.5-1.5 mm.sup.3; (4) culturing the small pieces treated in step (3) with an olfactory progenitor cell culture medium, and allowing them to grow adherently; wherein the olfactory progenitor cell culture medium comprises the following components: basic culture medium DMEM/DF12, epidermal cell growth factor (EGF), fibroblast growth factor (FGF), N2 cell culture supplement, B27 cell culture supplement, bovine serum albumin (BSA), glutamine and NEAA cell culture supplement; and (5) harvesting single cells when the cells grow adherently to a confluence of 75%-90%, adding the olfactory progenitor cell culture medium to prepare a cell suspension, and obtaining the olfactory progenitor cells; wherein the above operations are all performed under a strict aseptic condition.

2. The preparation method according to claim 1, wherein in step (1), the olfactory mucosa tissues between the upper turbinate and middle turbinate of a donor are taken with a specimen tissue forcep, and the obtained nasal mucosa tissue blocks are placed into an ice box.

3. The preparation method according to claim 1 or 2, wherein in step (3), the method further comprises the following steps of: preparing the small pieces into little pieces with a size of 0.2-0.4 mm.sup.3, and repeatedly rinsing the little pieces with the olfactory progenitor cell culture medium containing 160 U/mL gentamicin; preferably, the method comprises the following steps of: placing the small pieces into the olfactory progenitor cell culture medium containing 160 U/mL gentamicin, rinsing repeatedly, and tearing them into little pieces with a size of 0.2-0.4 mm.sup.3 with an ophthalmic tissue forcep; collecting the little pieces into a centrifuge tube and centrifuging at a centrifuge rotational speed of 800-2100 rpm for 10-30 minutes, and discarding the supernatant; re-adding the olfactory progenitor cell culture medium containing 160 U/mL gentamicin, and oscillating the precipitated tissue blocks sufficiently to make them loosen; and centrifuging again at a centrifuge rotational speed of 800-2100 rpm for 10-30 minutes.

4. The preparation method according to any one of claims 1-3, wherein in step (4), the culture device is wetted with a serum stock solution before the cell culture; preferably, in step (4), the culture are protected from light and performed under a condition of 37° C. and 5% CO.sub.2.

5. The preparation method according to any one of claims 1-4, wherein in step (4), the olfactory progenitor cell culture medium comprises the following components: 90 mL DMEM/DF12, 80 mg EGF, 60 mg FGF, 2 mL 1% N2 cell culture supplement, 3 mL 2% B27 cell culture supplement, 150 mg BSA, 0.2 mmol/mL glutamine, and 2 mL NEAA cell culture supplement; preferably, the olfactory progenitor cell culture medium further comprises the following components: 5-20 μmon P63 or P53 inhibitor, Pifithrin-α hydrobromide or Pifithrin-μ; or 1-10 μmol/L P63 or P53 agonist, PRIMA-1Met/Tenovin-1.

6. The preparation method according to any one of claims 1-5, wherein in steps (4) and step (5), the olfactory progenitor cell culture medium is changed every three days.

7. The preparation method according to any one of claims 1-6, wherein in step (4), when the cells grow adherently to a confluence of 55%-85%, the small pieces are collected and transferred into a new culture device for repeated culture; preferably, the small pieces are repeatedly used for 5-7 times to prepare 5-7 batches of olfactory progenitor cells.

8. The preparation method according to any one of claims 1-7, wherein in step (5), harvesting the single cells comprising the following steps of: rinsing the culture device with a DMEM/F12 solution, detaching the adherent single cells with an aqueous solution of 0.05% trypsin and an aqueous solution of 0.2 mmol/L EDTA, centrifuging to collect the single cells, and adding the olfactory progenitor cell culture medium to prepare a cell suspension; preferably, the cell density of the single cell suspension is 1×10.sup.5/mL-1×10.sup.6/mL.

9. The preparation method according to any one of claims 1-8, wherein in step (5), the olfactory progenitor cells are passaged every 2-3 days and subcultured for 1-3 generations.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0050] FIG. 1 shows the morphology of olfactory progenitor cells prepared according to the method of the present invention.

BEST MODES FOR CARRYING OUT THE INVENTION

[0051] The present invention will be described in detail below in conjunction with the examples, but is not limited to the following examples. Within the scope of knowledge possessed by those skilled in the art, various changes to the culture medium, cryopreservation protection solution, primary culture time, passage time, multi-batch harvest, and etc. can also be made without departing from the spirit of the present invention.

Example 1 Preparation of Olfactory Progenitor Cells According to the Method of the Present Invention

[0052] An olfactory progenitor cell culture medium was prepared by using 90 mL DMEM/DF12 (11320 Gibco), 80 mg EGF (Pepro Tech)), 60 mg FGF (Pepro Tech), 2 mL 1% N2 cell culture supplement (Gibco 17502-048), 3 mL 2% B27 cell culture supplement (gibco 17504-044), 150 mg BSA (Sigma), 0.2 mmol/mL glutamine (Gibco) and 2 mL NEAA cell culture supplement (100X, 11140050); and then culture flasks were coated with a serum stock solution (Gibco16000044) for 24 hours in advance.

[0053] Specifically, the method included the following steps of:

[0054] (1) obtaining the nasal olfactory mucosa: the blocks of olfactory mucosa tissue between the upper turbinate and middle turbinate of a donor were taken with a specimen tissue forcep, and the obtained blocks of nasal mucosa tissue were placed into a glass dish on an ice box and sent to a culture room immediately for aseptic processing;

[0055] (2) the obtained blocks of olfactory mucosa tissue were pretreated with 1 time volume of 5% (g/100 mL) trypsin aqueous solution and 1 time volume of 0.2 mmol/L EDTA aqueous solution at 36° C. for 15 minutes;

[0056] (3) the loose layers of surface and base membranes of the olfactory mucosa tissue blocks were removed under a dissecting microscope, and the olfactory mucosa tissue blocks were rinsed with PBS for 3 times and cut into small pieces with a size of 1 mm.sup.3;

[0057] the small pieces were placed into the olfactory progenitor cell culture medium containing 160 U/mL gentamicin, rinsed repeatedly for 3 times, and torn into little pieces with a size of about 0.4 mm.sup.3 with an ophthalmic tissue forcep; the tissue blocks were collected into a 10 mL centrifuge tube and centrifuged at a high speed with a centrifuge rotational speed of 1100 rpm for 20 minutes, and the supernatant was discarded; the olfactory progenitor cell culture medium containing 160 U/mL gentamicin was re-added, and the precipitated tissue blocks were oscillated sufficiently to make them loosen and then centrifuged at a high speed again with a centrifuge rotational speed of 1100 rpm for 20 minutes;

[0058] (4) the tissue blocks were added into an appropriate amount of culture medium and mixed uniformly, seeded into the pre-coated culture flasks to allow them grow adherently with a distance among the individual tissue blocks being about 4 mm-6 mm, and cultured in an incubator protected from light under 5% CO.sub.2 at 37° C. for 3 days; this step was used for the purpose of maximizing the removal of fibroblasts and hematopoietic cells;

[0059] (5) the culture flasks were gently shaken to collect the tissue blocks after 3 days of culture, and added with an appropriate amount of the olfactory progenitor cell culture medium; the tissue blocks were seeded into the pre-coated culture flasks with a distance among the individual tissue blocks being about 4 mm-6 mm, and cultured in an incubator protected from light under 5% CO.sub.2 at 37° C.; the obtained single cells were cultured continuously, and a half of the olfactory progenitor cell culture medium was changed every 3 days; on the 3rd day, the growth of multi-polar cells, such as bipolar and tripolar cells was observed under a microscope;

[0060] the culture flasks were gently shaken to collect the tissue blocks when the single cells grew to a confluence of about 60%; the tissue blocks were seeded into the pre-coated culture flasks with an appropriate distance among the individual tissue blocks being about 4 mm-6 mm, and cultured in an incubator protected from light under 5% CO.sub.2 at 37° C.; the adherent single cells in the original cell culture flasks were continued to be cultured by continuously adding the olfactory progenitor cell culture medium;

[0061] (6) when the single cells grow to a confluence of 75%-90%, the single cells were harvested by gently rinsing the culture flasks with a DMEM/F12 solution repeatedly for 2 times, detaching the adherent single cells with an aqueous solution containing 0.05% trypsin/0.2 mmol/L EDTA, and centrifuging at a rotational speed of 1100 rpm for 20 minutes; the olfactory progenitor cell culture medium was added to prepare a cell suspension which was divided into 3 parts depending on the cell number, transferred into the pre-coated culture flasks and cultured in an incubator protected from light under 5% CO.sub.2 at 37° C.;

[0062] wherein in steps (5) and (6), an appropriate amount of P63 or P53 inhibitor or P63 or P53 agonist was added depending on the observed culture situation; when the cells grew slowly, an appropriate amount of P63 or P53 agonist was added; when the cells grow too fast, an appropriate amount of P63 or P53 inhibitor was added, to make sure that the single cells can be passaged once every 2-3 days and that the primary cells can be isolated from the tissue blocks in 3-5 days;

[0063] steps (5) and (6) were repeated, and the tissue blocks were discarded when they grew and were used repeatedly for 5-7 times; 5-7 batches of primary olfactory progenitor cells can be obtained, and each batch of olfactory progenitor cells were subjected to subculture at a ratio of 1:3 repeatedly, which allows the cells to expand for 10-11 generations, and can obtain a large quantity of olfactory progenitor cells with a high purity.

Example 2 Preparation of Olfactory Progenitor Cells According to the Method of the Present Invention

[0064] An olfactory progenitor cell culture medium was prepared by using 90 ml DMEM/DF12 (11320 Gibco), 80 mg EGF (Pepro Tech), 60 mg FGF (Pepro Tech), 2 mL 1% N2 cell culture supplement (gibco), 3 mL 2% B27 cell culture supplement (gibco), 150 mg BSA (Sigma), 0.2 mmol/ml glutamine (Gibco), and 2 ml non-essential amino acids (11140050 GIBCO); and then the culture flasks were coated with serum (Gibco 16000044) for 24 hours in advance.

[0065] Specifically, the method included the following steps of:

[0066] (1) obtaining the nasal olfactory mucosa: the blocks of olfactory mucosa tissue between the upper turbinate and middle turbinate of a donor were taken with a specimen tissue forcep, and the obtained blocks of nasal mucosa tissue were placed into a glass dish on an ice box and sent to a culture room immediately for aseptic processing;

[0067] (2) the obtained blocks of olfactory mucosa tissue were treated with 1 time volume of 5% (g/100 mL) trypsin and 1 time volume of 0.2 mmol/L EDTA at 38° C. for 15 minutes;

[0068] (3) the loose layers of surface and base membranes of the olfactory mucosa tissue blocks were removed under a dissecting microscope, and the olfactory mucosa tissue blocks were rinsed with PBS for 3 times and cut into small pieces with a size of 1 mm.sup.3;

[0069] the small pieces were placed into the olfactory progenitor cell culture medium containing 160 U/mL gentamicin, rinsed repeatedly for 3 times, and torn into little pieces with a size of about 0.4 mm.sup.3 with an ophthalmic tissue forcep; the tissue blocks were collected into a 10 mL centrifuge tube and centrifuged at a high speed with a centrifuge rotational speed of 2100 rpm for 10 minutes, and the supernatant was discarded; the olfactory progenitor cell culture medium containing 160 U/mL gentamicin was re-added, and the precipitated tissue blocks were oscillated sufficiently to make them loosen, and then centrifuged at a high speed again with a centrifuge rotational speed of 2100 rpm for 30 minutes;

[0070] (4) the tissue blocks were added into an appropriate amount of culture medium and mixed uniformly, seeded into the pre-coated culture flasks to allow them to grow adherently with an appropriate distance among the individual tissue blocks being about 4 mm-6 mm, and cultured in an incubator protected from light under 5% CO.sub.2 at 37° C. for 3 days; this step was used for the purpose of maximizing the removal of fibroblasts and hematopoietic cells;

[0071] (5) the culture flasks were gently shaken to collect the tissue blocks after 3 days of culture, and added with an appropriate amount of olfactory progenitor cell culture medium; the tissue blocks were seeded into the pre-coated culture flasks with an appropriate distance among the individual tissue blocks being about 4 mm-6 mm, and cultured in an incubator protected from light under 5% CO.sub.2 at 37° C.;

[0072] the obtained single cells were cultured continuously, and a half of olfactory progenitor cell culture medium was changed every 3 days; on the 5th day, the growth of multi-polar cells, such as bipolar and tripolar cells was observed under a microscope;

[0073] the culture flasks were gently shaken to collect the tissue blocks when the single cells grew to a confluence of about 85%; the tissue blocks were seeded into the pre-coated culture flasks with an appropriate distance among the individual tissue blocks being about 4 mm-6 mm, and cultured in an incubator protected from light under 5% CO.sub.2 at 37° C.; the adherent single cells in the original cell culture flasks were continued to be cultured by continuously adding the olfactory progenitor cell culture medium;

[0074] (6) the single cells were harvested when the cells grew to a confluence of 75%-90% by gently rinsing the culture flasks with a DMEM/F12 solution repeatedly for 2 times, detaching the adherent single cells with an aqueous solution containing 0.05% trypsin/0.2 mmol/L EDTA, and centrifuging at a rotational speed of 2100 rpm for 5 minutes; the olfactory progenitor cell culture medium was added to prepare a cell suspension, which was divided into 3 parts according to the cell number, transferred into the pre-coated culture flasks and cultured in an incubator protected from light under 5% CO.sub.2 at 37° C.;

[0075] in steps (5) and (6), an appropriate amount of P63 or P53 inhibitor or P63 or P53 agonist was added according to the observed culture situation; when the cells grew slowly, an appropriate amount of P63 or P53 agonist was added; when the cells grew too fast, an appropriate amount of P63 or P53 inhibitor was added, to make sure that the single cells can be passaged once every 2-3 days and that the primary cells can be isolated from the tissue blocks in 3-5 days.

[0076] steps (5) and (6) were repeated, and the tissue blocks were discarded when they grew and were used repeatedly for 5-7 times; 5-7 batches of primary olfactory progenitor cells can be obtained, and each batch of olfactory progenitor cells were subjected to subculture at a ratio of 1:3 repeatedly, which allows the cells to expand for 10-11 generations, and can obtain a large quantity of olfactory progenitor cells with a high purity.

Example 3 Identification of Olfactory Progenitor Cells Obtained According to the Method of the Invention

[0077] The single cells obtained by culture were identified by conventional immunohistochemical staining, and the results are shown in FIG. 1. Cytological identification of olfactory progenitor cells (with nestin, GFAP, and Tuj-1 or OMP cytochemical staining) shows that most cells are nestin positive, while some of them are also GFAP positive and some of them are also Tuj-1 positive, and there are no cells that are only nestin positive, but not positive for other markers for costaining, which are consistent with the characteristics of olfactory progenitor cells.

Example 4 Preparation of Olfactory Ensheathing Cells or Olfactory Neuron Cells with the Olfactory Progenitor Cells Obtained According to the Method of the Invention

[0078] The single cells obtained by culture differentiated directionally to into olfactory ensheathing cells or olfactory neuron cells using the techniques described in Chinese invention patent Nos. 201510935540.3 or 201510516055.2. The cells are used for clinical treatment according to the Clinical Application Guidelines of the International Association of Neurorestoratology.

Example 5 Application of Cells Prepared by the Present Application

[0079] Case 1: an American male with a complete thoracic spinal cord (T6) injury for more than nine years had insisted on regular rehabilitation training locally without any recovery of neurological function. He received the treatment with our olfactory ensheathing cells in 2005. After two weeks of treatment, his sensory level decreased and the flexor-extensor strength of hip and knees partially recovered. After returning to the United States, the neurological function continued to improve and recover, and bladder or bowel control partially recovered. Before the treatment, the ASIA score was 50 for motor, 52 for pin prick sensation, and 52 for soft touch. A follow-up after half a year of treatment showed the ASIA score was 56 for motor, 60 for pin prick sensation, and 60 for soft touch sensation. The function index of spinal cord injury according to the International Association of Neurorestoratology was 21 before the treatment and 29 at time of the follow-up after half a year of treatment. The U.S. Fox TV reported that “a patient with an advanced complete spinal cord injury stands up and walks again”.

[0080] Case 2: an American male was diagnosed with motor neuron disease (amyotrophic lateral sclerosis), and then his condition gradually deteriorated. He staggered with weak strength of upper limbs and hands, and had difficulty in lifting objects. His life expectancy was determined to be only about two years. After receiving our treatment with olfactory ensheathing cell in 2005, the strength of upper and lower limbs and hands increased, and the walk returned to normal. After returning to the United States, he resumed playing golf (he lost the ability to play golf half a year before the treatment with olfactory ensheathing cell), and the therapeutic improvement and function had been maintained for one and a half years. He came back to receive the treatment with olfactory ensheathing cell again when it was difficult for him to play golf again. Then, he received the treatment with olfactory ensheathing cell every one and a half years to two years, and a continued follow-up reported that he was still alive in 2016.

[0081] Case 3: an Italian female had suffered from sequelae of cerebral apoplexy in the left side for three years. She can only stand with the aid of a brace on her right ankle, and walk in a claudication gait. She received our treatment with olfactory ensheathing cells in 2006 and then made an gradual improvement of neurological function. A follow-up after two years reported that she did not require a brace for walking, and her gait returned to normal. The Barthel index was 85 before the treatment, and 100 at the time of the follow-up two years later.

[0082] The above is a specific description of the present invention in combination with the examples, but the present invention is not limited to the above examples. Within the scope of knowledge possessed by those skilled in the art, various changes can also be made without departing from the spirit of the present invention, and direct or indirect applications of the present invention in other related technical fields are all included in the patent protecting scope of the present invention for the same purpose.