Poultry Egg-Based Culture Medium

Abstract

Disclosed is a poultry egg-based culture medium relating to the field of biotechnology, in particular a culture medium that can be used for cultivating mammalian cells, the culture medium containing a poultry egg content and a diluent. Also related is preparation and use of the culture medium, and a method of using the culture medium for cultivating mammalian cells.

Claims

1. A culture medium for cultivating mammalian cells, the culture medium comprising: a poultry egg content; and a diluent: wherein the poultry egg content has a volume percentage ranging from 0.1% to 99% in the culture medium.

2. The culture medium according to claim 1, wherein the culture medium has one or more of the following characteristics: the poultry egg content is egg yolk and/or egg white of a poultry egg; the poultry egg is selected from the group consisting of hen egg, duck egg, goose egg, ostrich egg, quail egg, other poultry egg, or any combination thereof; the poultry egg is unfrozen, or thawed after being frozen; the diluent is phosphate-buffered saline, Ringer's solution, Hank's Balanced Salt Solution, or HEPES buffer; the culture medium does not contain an antibiotic (e.g., ampicillin-streptomycin, or a mixed solution of penicillin, streptomycin and amphotericin B); a pH indicator, preferably phenol red; an energy source, such as a carbohydrate, preferably, glucose; an amino acid; a vitamin; and/or another organic compound required in a low concentration; a trace element; an inorganic salt (e.g., sodium bicarbonate); or any combination thereof.

3. The culture medium according to claim 1, wherein the poultry egg content is egg yolk and/or egg white.

4. The culture medium according to claim 1, wherein the culture medium does not contain a fetal bovine serum.

5. A method of us of the culture medium according to claim 1 comprising: cultivating mammalian cells; wherein, the cultivating is adherent cultivating, suspension cultivating or immobilization cultivating.

6. The method according to claim 5, wherein the culture medium is mixed with another culture medium that is applicable to cultivating mammalian cells, including but not limited to mixing with RPMI-1640, DMEM, MEM, M199, F-10, F-12, DMEM/F-12 1:1 or McCoy's 5A medium.

7. The method according to claim 5, wherein the mammalian cells have one or more of the following characteristics: the mammalian cells are derived from a bovine, equine, swine, canine, feline, rodent, or primate; the mammalian cells are derived from a connective tissue, muscle tissue, nerve tissue, or epithelial tissue; the mammalian cells are derived from heart, bladder, ovary, pancreas, breast, liver, lung, stomach, kidney, intestine, skin, bone, muscle, esophagus, trachea, male reproductive system, female reproductive system, lymph, nerve, or thyroid; the mammalian cells are non-tumor cells or tumor cells; the mammalian cells are cells of single type, or a mixture of cells of multiple types.

8. A method for preparing a culture medium the method comprising the following steps: obtaining a poultry egg content in an aseptic condition; and diluting the poultry egg content with a diluent.

9. A method for cultivating mammalian cells, the method comprising using the culture medium according to claim 1, wherein the cultivating is adherent cultivating, suspension cultivating or immobilization cultivating.

10. The method according to claim 9, the method comprising: providing a culture medium comprising a poultry egg content and a diluent, the poultry egg content having a volume percentage ranging from 0.1% to 99% in the culture medium; and mixing the culture medium with another medium that is applicable to cultivating mammalian cell, e.g. mixing with RPMI-1640, DMEM, MEM, M199, F-10, F-12, DMEM/F-12 1:1 or McCoy's 5A medium.

11. The culture medium according to claim 1, wherein the culture medium has one or more of the following characteristics: the poultry egg content is egg yolk and/or egg white of a poultry egg; the poultry egg is selected from the group consisting of hen egg, duck egg, goose egg, ostrich egg, quail egg, other poultry egg, or any combination thereof; the poultry egg is unfrozen, or thawed after being frozen; the diluent is phosphate-buffered saline, Ringer's solution, Hank's Balanced Salt Solution, or HEPES buffer; the culture medium contains an antibiotic (e.g., ampicillin-streptomycin, or a mixed solution of penicillin, streptomycin and amphotericin B); a pH indicator, preferably phenol red; an energy source, such as a carbohydrate, preferably, glucose; an amino acid; a vitamin; and/or another organic compound required in a low concentration; a trace element; an inorganic salt (e.g., sodium bicarbonate); or any combination thereof.

12. The culture medium according to claim 1, wherein the culture medium contains a fetal bovine serum.

13. The method for preparing the culture medium according to claim 8, further comprising: adding an antibiotic, phenol red, glucose, vitamin, inorganic salt, fetal bovine serum, or any combination thereof to the poultry egg content.

14. The culture medium according to claim 3, wherein the culture medium does not contain an antibiotic.

15. The culture medium according to claim 3, wherein the egg yolk and egg white are derived from poultry eggs of the same type or different types.

16. The culture medium according to claim 3, wherein in the poultry egg content, the ratio of egg yolk to egg white is 99:1 to 1:99.

Description

SPECIFIC MODELS FOR CARRYING OUT THE PRESENT INVENTION

[0033] The present invention will be further described in detail below in conjunction with specific embodiments. However, it should be pointed out that the following examples are only for illustrating the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples, unless otherwise specified, were all conventional methods. The materials, reagents, instruments, etc. used in the following examples were obtained from commercial sources unless otherwise specified. For the quantitative experiments in the following examples, each experiment was repeated three times, and the averages of results were taken as final results.

Example 1

Culture of Different Cell Lines in Hen Egg-Based Culture Medium

[0034] (1) Preparation of culture medium without antibiotics and glucose: fresh hen eggs were taken, their eggshells were opened in aseptic manipulation, their egg white and egg yolk were taken out and weighed, the volume X thereof was calculated using density of 1 g/ml, phenol red (final concentration: 0.0159 g/L) and 0.15M of NaHCO.sub.3 (2.933X) were added, and the final volume was adjusted to 10× (diluted 10 times) with PBS. The prepared culture medium was stored at 4° C. for no more than one week.

[0035] (2) Preparation of culture medium with antibiotics and glucose: fresh hen eggs were taken, their eggshells were opened in aseptic manipulation, their egg white and egg yolk were taken out and weighed, the volume X thereof was calculated using density of 1 g/mL, phenol red (final concentration: 0.0159 g/L), 0.15M of NaHCO.sub.3 (2.933X), glucose (final concentration: 4.5g/L), ampicillin-streptomycin (final concentrations: 100 units/mL and 100 μg/mL, respectively) were added, and the final volume was adjusted with PBS to 10× (diluted 10 times). The prepared culture medium was stored at 4° C. for no more than one week.

[0036] (3) The culture medium prepared in step (1) or (2) was used to directly cultivate cells in conventional conditions (37° C., 5% CO.sub.2). For adherent culture, after trypsin digestion, cells were cultivated in a conventional medium (DMEM or RPMI medium, both containing 10% FBS and antibiotics) for 4 to 6 hours to make them adherent, then the DMEM or RPMI medium was then replaced with the poultry egg-based medium. For suspension culture, cells were directly cultivated with the poultry egg-based culture medium. The culture medium was replaced every 1 to 3 days.

[0037] (4) A conventional culture medium (DMEM or RPMI medium, both containing 10% FBS and antibiotics) and a serum-free medium (DMEM or RPMI medium, both containing antibiotics only) were used as controls.

[0038] The inventors of the present invention compared the cell proliferation folds of different cell lines cultivated in the hen egg-based culture media of the present invention and the conventional culture media (with antibiotics added) for 6 days (a result less than 1 indicates a decrease in the number of cells). The results were shown in Table 1.

TABLE-US-00001 TABLE 1 Hen egg- based Hen egg- culture Conventional Conventional based culture medium culture culture medium (not medium medium (not (containing containing (containing containing antibiotics antibiotics Cell line Cell type Species 10% FBS) FBS) and glucose) or glucose) 293A Human Non-tumor Human 12.40 5.00 8.33 7.50 embryonic adherent kidney cell cell 293T Human Non-tumor Human 6.80 2.00 4.20 5.40 embryonic adherent kidney cell cell H9 Human T Non-tumor Human 7.21 1.80 3.56 2.43 lymphocyte suspension line cell HeLa Human Tumor Human 9.61 2.84 5.00 5.38 cervical adherent cancer cell cell 4T1 Mouse Tumor Mouse 5.46 0.36 3.00 3.62 breast cancer adherent cell cell CT26 Mouse Tumor Mouse 18.96 0.08 1.70 2.88 colon cancer adherent cell cell GC2-spd Normal Mouse 12.00 6.00 8.00 5.60 Mouse adherent spermatocyte cell Ins-1 Rat islet Tumor Rat 10.40 2.80 6.40 6.80 cell tumor adherent cell cell

[0039] It could be seen from Table 1 that the hen egg-based culture medium could meet the requirements for growth and proliferation of a variety of mammalian cells and could be used to replace a conventional medium.

Example 2

Culture of Different Cell Lines in Duck Egg-Based Medium

[0040] With reference to the method of Example 1, duck egg-based culture medium without antibiotics and glucose was prepared on the basis of fresh duck eggs and was used in cell culture.

[0041] Table 2 showed the cell proliferation folds of different cell lines after being cultivated for 6 days in the duck egg-based culture medium.

TABLE-US-00002 TABLE 2 Duck egg-based culture medium (not containing Cell line Cell type Species antibiotics or glucose) 293A Human Non-tumor Human 6.27 embryonic adherent cell kidney cell HeLa Human Tumor Human 3.50 cervical adherent cell cancer cell GC2 Mouse Normal Mouse 6.62 spermatocyte adherent cell CT26 Mouse Tumor Mouse 3.42 colon cancer adherent cell cell

[0042] It could be seen from Table 2 that the culture medium based on duck eggs could meet the growth and proliferation requirements of a variety of mammalian cells and could be used to replace a conventional media.

Example 3

Culture of Different Cell Lines in Medium Prepared Based on Frozen-Thawed Poultry Eggs

[0043] With reference to the method of Example 1, frozen hen eggs or duck eggs (frozen at −20° C. for 1 month) were thawed to prepare a medium without antibiotics and glucose, and a variety of cells were cultivated.

[0044] Table 3 showed the cell proliferation folds of different cell lines after being cultivated for 6 days in a medium prepared with thawed eggs or duck eggs.

TABLE-US-00003 TABLE 3 Hen Duck egg-based egg-based culture culture medium medium Cell line Cell type Species (frozen) (frozen) 293A Human Non-tumor Human 8.60 6.10 embryonic adherent Cell kidney cell HeLa Human Tumor Human 4.52 4.21 cervical adherent cell cancer cell GC2 Mouse Normal Mouse 6.53 2.13 spermatocyte adherent cell CT26 Mouse Tumor Mouse 5.46 3.30 colon cancer adherent cell cell

[0045] It could be seen from Table 3 that the culture medium prepared based on frozen-thawed poultry eggs could meet the requirements for growth and proliferation of a variety of mammalian cells.

Example 4

Influences of Egg White-Egg Yolk Ratio and Dilution Ratio on the Effect of Cultivating 293A Cells with Egg-Based Culture Medium

[0046] With reference to the method of Example 1, a fresh hen egg-based culture medium without antibiotics and glucose was prepared, and the egg white-egg yolk ratio and the dilution ratio were changed, and then cells were cultivated. The results were shown in Table 4.

TABLE-US-00004 TABLE 4 Egg Cell white-egg Dilution proliferation yolk ratio ratio folds Normal (64.7:35.3) 1:20 10.15 Normal (64.7:35.3) 1:50 5.72 Normal (64.7:35.3)  1:100 4.43  0:100 1:10 2.57  1:99 1:10 4.29  2:98 1:10 7.14  5:95 1:10 7.14 10:90 1:10 4.26 25:75 1:10 6.40 50:50 1:10 4:38 75:25 1:10 2.76 90:10 1:10 1.80 Note: Dilution was performed using phosphate buffer.

Example 5

Influences of Other Factors on the Effect of Cultivating 293 Cells with Egg-Based Culture Medium

[0047] With reference to the method of Example 1, a fresh hen egg-based culture medium was prepared, and other factors were changed, and then cells were cultivated. The results were shown in Table 5.

TABLE-US-00005 TABLE 5 Other Cell Buffer/ substance proliferation Purpose solution added folds Adding glucose PBS Glucose, 2 g/L 17.15 Adding regular medium PBS 1% DMEM* 2.00 Adding regular medium PBS 10% DMEM* 5.43 Adding regular medium PBS 25% DMEM* 7.71 Adding FBS PBS 1% FBS 4.14 Different buffer Earle's None 6.50 buffer Different buffer Ringer's 1X PSA** 4.00 solution Different buffer Hank's 1X PSA** 8.71 Balanced Salt Solution Note: *DMEM medium was a high-glucose DMEM medium supplemented with 10% FBS and antibiotics (1XPSA); **1X PSA: penicillin-streptomycin-amphotericin B mixed solution: penicillin 100 U/mL, chloramphenicol 0.1 mg/mL, amphotericin B 0.25 ug/mL.

[0048] The above descriptions are only the preferred examples of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, and so on made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.