Preparation method for olfactory ensheathing cells

Abstract

Provided is a preparation method for olfactory ensheathing cells. The method comprises the steps of formulation of a cell medium, collection and pretreatment of tissues, enzymatic digestion, cell culture, cryopreservation, and differentiation culture. The prepared olfactory ensheathing cells can maintain the proliferative capacity for a long time, and still possess the activity of olfactory ensheathing cells after 11.sup.th passage.

Claims

1. A preparation method for olfactory ensheathing cells, comprising the following steps: (1) culturing single olfactory ensheathing cells derived from the olfactory mucosa of upper and middle turbinate in a culture medium for olfactory ensheathing cells under the conditions of 37 C. and 5% CO.sub.2, and allowing the single olfactory ensheathing cells to grow adherently; (2) mixing the cells obtained by filtering a culture supernatant obtained from step (1) with a culture medium for olfactory ensheathing cells, culturing under the conditions of 37 C. and 5% CO.sub.2, and allowing the cells to grow adherently; and (3) passage culture of the cells, wherein the cells obtained still have the activities of olfactory ensheathing cells, after 11.sup.th passage, wherein the culture medium for olfactory ensheathing cells is DMEM/DF12 (Gibco) as a basic culture medium, supplemented with the neurotrophic factors comprising 20-60 ng/ml EGF, 20-80 ng/ml FGF, 1-2 ml N2 (100), 2-3 ml B27 (50) and 0.1 g/ml T3 (Sigma) at final concentrations.

2. The method according to claim 1, wherein the olfactory ensheathing cells are the olfactory ensheathing cells derived from human olfactory mucosa.

3. The method according to claim 1, wherein the preparation method for single olfactory ensheathing cells comprises the following step: digesting tissue blocks of the olfactory mucosa of upper and middle turbinate with an enzyme to obtain single cells.

4. The method according to claim 3, wherein the tissue blocks of the olfactory mucosa of upper and middle turbinate are washed before the digestion to remove the residual blood on the surface.

5. The method according to claim 1, wherein the innoculation density of the culture in step (1) is 110.sup.4 cells/ml.

6. The method according to claim 1, wherein the preparation method further comprises a step of differentiation culture of the olfactory ensheathing cells.

7. The method according to claim 1, wherein the preparation method comprises the following steps: Step 1. formulating a culture medium for olfactory ensheathing cell with neurotrophic factors; Step 2. collection of tissues: picking up an appropriate amount of the olfactory mucosa tissues of upper and middle turbinate under local anesthesia; Step 3. pretreatment of tissue blocks: washing the residual blood on the surface of the obtained tissue blocks and processing the tissue blocks into ones in a size of 1 mm.sup.3; Step 4. digestion by a combination of two enzymes: collecting the scissor-minced tissue blocks, adding 2 volumes of Collagenase I and a neutral protease, and digesting in water bath at 37 C. under vibration; Step 5. primary culture: collecting the single olfactory ensheathing cells obtained by the digestion and placing them in a centrifuge tube, mixing thoroughly with a culture medium for olfactory ensheathing cells, then inoculating in a cell culture flask, and culturing in an incubator at 37 C. and 5% CO.sub.2; Step 6. passage culture: collecting a culture supernatant of the cells to be passaged when the cells grow adherently to around 90%, filtering and mixing the supernatant with a culture medium for olfactory ensheathing cells in a ratio of 1:3, to formulate a culture medium for the present passage; Step 7. cryopreservation at an ultralow temperature: collecting and filtering a supernatant of cells to be cryopreserved when the cells grow adherently to around 90%, to formulate a cryopreservation medium specific for the olfactory ensheathing cells consisting of the autologous serum, the cell culture supernatant, DMEM/F12 and DMSO; and cryopreserving according to the routine procedures; and Step 8. differentiation culture: adding a formulated culture medium for the differentiation of glial cells, wherein the culture medium for olfactory ensheathing cells: the autologous serum=87:13, and culturing for differentiation according to the routine procedures.

8. The method according to claim 1, wherein the preparation method for single olfactory ensheathing cells comprises the following step: digesting tissue blocks of the olfactory mucosa of upper and middle turbinate with Collagenase I and a neutral protease to obtain single cells.

9. The method according to claim 8, wherein the neutral protease is Dispase II for tissues.

10. The method according to claim 1, wherein the preparation method for single olfactory ensheathing cells comprises the following step: digesting tissue blocks of the olfactory mucosa of upper and middle turbinate in a size of 1 mm.sup.3 with Collagenase I and a neutral protease in water bath at 37 C. under vibration to obtain single cells, wherein the ratio by volume of Collagenase I and a neutral protease to the tissue blocks of the olfactory mucosa of upper and middle turbinate is 2:1.

11. The method according to claim 4, wherein the residual blood on the surface is removed by a solution of penicillin and a physiological saline in a ratio of 1:1.

12. The method according to claim 4, further comprising a step of cutting the olfactory mucosa into tissue blocks in a size of 1 mm.sup.3 in a culture dish with a sterile scissor.

13. The method according to claim 5, wherein the cells grow adherently to 90%.

14. The method according to claim 1, wherein during the passage culture, the culture supernatant of the cells to be passaged is collected, filtrated and mixed with a culture medium for olfactory ensheathing cells from olfactory mucosa in a ratio of 1:3 to formulate a culture medium for passage.

15. The method according to claim 10, wherein Collagenase I is used at a concentration of 0.1% and the neutral protease is used at a concentration of 0.2%.

16. The method according to claim 1, wherein the innoculation density of the culture in step (2) is 110.sup.4 cells/ml.

17. The method according to claim 16, wherein the cells grow adherently to 90%.

18. The method according to claim 1, wherein the preparation method further comprises a step of cryopreservation of the olfactory ensheathing cells.

19. The method according to claim 18, wherein the cryopreservation is cryopreservation at an ultralow temperature.

20. The method according to claim 19, wherein the cryopreservation of the olfactory ensheathing cells at an ultralow temperature comprises the following steps: filtering the culture supernatant when the cells in step (2) grow adherently to 90% to prepare a cryopreservation medium for the cryopreservation of olfactory ensheathing cells.

21. The method according to claim 20, wherein the cryopreservation medium for olfactory ensheathing cells consists of the autologous serum, the culture supernatant, DMEM/F12 and DMSO.

22. The method according to claim 20, wherein the cryopreservation medium for olfactory ensheathing cells consists of the autologous serum, the culture supernatant, DMEM/F12 and DMSO in a ratio by volume of 5:2:2:1.

23. The method according to claim 6, wherein the step of differentiation culture of the olfactory ensheathing cells comprises adding a culture medium for the differentiation of glial cells to the olfactory ensheathing cells and culturing for differentiation.

24. The method according to claim 23, wherein the culture medium for the differentiation of glial cells consists of a culture medium for olfactory ensheathing cells and the autologous serum in a ratio by volume of 87:13.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the results of microscopy examination on the olfactory ensheathing cells, magnification: 10/0.25.

(2) FIG. 2 shows the confocal microscopy identifications of the olfactory ensheathing cells using immunohistochemical staining, magnification: 20/0.5.

DETAIL DESCRIPTION

(3) 1. Formulation of a Culture Medium for Olfactory Ensheathing Cells

(4) A culture medium for olfactory ensheathing cells was formulated with DMEM/DF12 (Gibco) as a basic culture medium, which was supplemented with the culture factors comprising 20-60 ng/ml EGF, 20-80 ng/ml FGF, 1-2 ml N2 (100), 2-3 ml B27 (50) and 0.1 g/ml T3 (Sigma) at final concentrations, then filtered and sterilized in a clean bench and stored at 4 C. for later use.

(5) 2. Collection of Tissues

(6) Two days before the acquisition of tissue blocks, the nasal cavity was cleaned up and confirmed to be uninfected. Before the acquisition, two operations of local anaesthesia were performed intranasally by inserting a sheet of cotton yarn dipped with a physiological saline containing 1.2%-1.5% tetracaine into the nasal cavity with a gun-shaped forceps, each being performed for 5-10 min; the olfactory mucosa tissues close to the upper and outer of upper and middle turbinate were taken with forceps for ethmoidal sinus, and the obtained tissues were placed into a glass dish; and then the glass dish was placed into an ice box, which was taken back to a culture room immediately.

(7) 3. Pretreatment of Tissues

(8) After being taken back to the lab, the tissue blocks were washed with a physiological saline containing 100 U/ml penicillin to remove the residual blood on the surface of the mucosa tissue blocks, and cut into ones in a size of 1 mm.sup.3 with a sterile ophthalmological scissors in a glass dish.

(9) 4. Digestion by a Combination of Two Enzymes

(10) The scissor-minced tissue blocks were collected, to which added 2 volumes of Collagenase I and a neutral protease, and digested in water bath at 37 C. under vibration for 15 min, and then were pipetted repeatedly with a pipette having a thick elbow, and allowed to settle naturally for 1 min. The supernatant was transferred into a centrifuge tube and the digestion was terminated. The above operation was repeated 3 times so that the tissues could be digested into a single cell suspension. All the cell suspensions after the terminatation of digestion were collected together and centrifuged at 1200 r/4 min, washed with DMEM/F12 and centrifuged at 1200 r/4 min for a total of 3 times.

(11) 5. Primary Culture

(12) A culture medium for olfactory ensheathing cells was added to prepare a single cell suspension, which was inoculated into a plastic culture flask with a bias opening treated by poly-L-lysine at a density of 110.sup.4 cells/ml, then cultured in a constant temperature and humidity incubator at 37 C. and 5% CO.sub.2.

(13) 6. Passage Culture

(14) The supernatant of culture was collected when the cells grew to around 90% confluence, filtered with a 0.22 m filter and mixed with a culture medium for olfactory ensheathing cells in a ratio of 1:3 to formulate the culture medium for the present passage; the cells was collected, to which added an appropriate amount of culture medium for passage to prepare a cell suspension, after staining with trypan blue, viable cells were counted under a microscope and then passaged again, with the density of the passaged cells being 110.sup.4 cells/cm.sup.3.

(15) 7. Cryopreservation at an Ultralow Temperature

(16) The supernatant of culture was collected when the cells grew to around 90% confluence, filtered with a 0.22 m filter to formulate a cryopreservation medium specific for olfactory ensheathing cells (a ratio by volume of the autologous serum:the supernatant of cell culture:DMEM/F12:DMSO=5:2:2:1); the cells for cryopreservation were collected routinely, to which added a cryoprotectant, and the final density of the cells was adjusted to 510.sup.6 cells/ml510.sup.7 cells/ml. The cells were stored at 4 C. for 60 minutes and at 20 C. for 70 minutes, and were cryopreserved at an ultralow temperature of 80 C. overnight before being placed in a liquid nitrogen tank.

(17) 8. Differentiation Culture

(18) A differentiation medium for glial cells (a ratio by volume of a culture medium for olfactory ensheathing cells: the autologous serum=87:13) was formulated, and preserved at 4 C. for later use; the formulated differentiation medium for glial cells was added and a differentiation culture was carried out according to the routine procedures.

(19) The present invention has been specifically described above with reference to the examples. However, the present invention is not limited to the above examples. Within the scope of the knowledge of those of ordinary skill in the art, various modifications may be made without departing from the spirit of the present invention, or the present invention can be applied directly or indirectly in other related technical fields, all of which are similarly included in the scope of the present invention.