Stable amorphous calcium carbonate as cell culture medium supplements

Abstract

The invention relates to a stable amorphous calcium carbonate as a cell culture medium supplement. A stable amorphous calcium carbonate (ACC) as a supplement for cell culture media and a cell culture media supplement comprising the stable ACC are provided. In particular, the stabilized ACC may be used to promote in vitro growth of cell and tissue cultures, gametes and embryos.

Description

[0001] This application is a divisional application of an application filed on January 17, 2017, with application number 201780017739.5 and invention name “Stable amorphous calcium carbonate as a cell culture medium supplement”. Field of the Invention

[0002] The present invention provides a stable amorphous calcium carbonate as a supplement for cell culture medium. In particular, ACC can be used to promote the in vitro growth of cells and tissue cultures. In another aspect of the present invention, a cell culture medium supplement comprising stable ACC is provided. Background of the Invention

[0004] Cell culture techniques allow animal, plant or insect cells removed from tissue to grow in vitro when the appropriate nutrients and conditions are provided. Cell culture techniques have many applications, including the study of cellular processes, the evaluation of the effects of various chemical compounds or drugs on specific cell types, the synthesis of valuable biological products on an industrial scale, and the generation of expanded cells for transplantation purposes. Cell culture techniques are used in in vitro fertilization, stem cell research, and vaccine production.

[0005] One of the most important uses of cell culture technology is the mass production of biological products, including specific proteins such as monoclonal antibodies. The number of such commercially valuable biological products has increased rapidly in the past few decades and has led to the current widespread interest in mammalian cell culture technology.

[0006] The composition of the culture medium for culturing cells is very important because it has an impact on cell survival, proliferation and the production of interested biological products. Many different cell culture media with different levels of specificity for cell culture have been developed. Some culture media are basal culture media, which can be supplemented according to the needs of different cell cultures, and others are more complex culture media. Hundreds of individual compounds can be added to the cell culture medium to obtain the desired effect. But the concept of culture medium supplementation is usually limited to the addition of substances, which usually promote the establishment and maintenance of cell culture. The most commonly used supplements are minerals, vitamins, amino acids, hormones and serum, the most common being fetal bovine serum, horse serum or human serum. However, since the use of serum may be undesirable in many cases, growth medium or serum-free medium with reduced serum content is used.

[0007] There is ongoing research and development of culture media and in particular of supplements to culture media that allow for the support and proliferation of cell and tissue cultures, in particular providing beneficial large scale production culture conditions. SUMMARY OF THE INVENTION

[0009] Surprisingly found that, compared with the cells grown in the culture medium supplemented with other calcium sources, the cell culture medium supplemented with stable amorphous calcium carbonate (ACC) provides the enhanced growth of various cells.Especially, observed that stable ACC promotes the formation of myotubes in mdx cells, and the mdx cells are the model of Duchenne muscular dystrophy.In addition, in the culture medium comprising stable ACC, observed the in vitro development of embryonic promotion, the culture medium is the single culture medium of "one step" or "continuous" culture medium (cleavage medium (Cleavage medium)).Surprisingly found that the embryo grown in the cleavage medium supplemented with stable ACC shows fast cleavage and higher hatching rate.In other cases, the neural cell grown in the culture medium supplemented with ACC shows enhanced neurofilament regeneration, and the stem cell grown in the culture medium supplemented with ACC shows faster amplification and differentiation. It was also surprisingly found that sperm incubated in the presence of ACC exhibited much higher motility after the swim-up procedure and the concentration of sperm cells (motile sperm) in the upper phase incubated with ACC was up to 7 times higher than that in the untreated samples. Furthermore, it has been shown that ACC has no biphasic effect on sperm motility, which is a finding that the addition of Ca to sperm samples 2+ All these observations suggest that stabilized ACC can be used as a supplement in cell culture media to promote and drive cell growth and provide better functionality.

[0010] On the one hand, the present invention provides a kind of cell culture medium, wherein the cell culture medium is supplemented with amorphous calcium carbonate (ACC) stabilized by at least one stabilizer, and wherein the cell culture medium is suitable for the growth of biological culture. According to some embodiments, the culture medium is suitable for the culture, tissue culture, organ culture or organ of growing cells. According to other embodiments, the cell culture medium supplemented with stable ACC can promote or promote growth, such as promoting the proliferation, maturation, reproduction, regeneration, development, preservation, such as cryopreservation and / or differentiation of cells, tissues and organs. According to one embodiment, the cell is an animal, plant or insect cell. According to one embodiment, the cell culture medium supplemented with stable ACC is suitable for the growth of cell culture, tissue culture, organ culture, and optionally can promote the growth of cell culture, tissue culture, organ culture, wherein the culture is a cell, tissue or organ culture of an animal, plant or insect. According to one embodiment, the cell culture medium supplemented with stable ACC is suitable for the growth of stem cells such as embryonic stem cells, chorionic stem cells, amniotic membrane stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, glial stem cells, nasal olfactory mucosa (NOM) stem cells, adult tissue-specific stem cells and induced pluripotent stem cells, or suitable for the growth of embryos such as human or non-human mammal embryos. In other embodiments, such a cell culture medium can promote stem cell proliferation, amplification and / or differentiation, can promote gamete maturation or can promote embryonic development. According to some embodiments, the cell culture medium supplemented with stable ACC is suitable for the growth of yeast or bacteria, and optionally can promote the growth of yeast or bacteria. In some embodiments, the bacteria are Escherichia coli (E.coli) or probiotics, such as bifidobacterium (Bifidobacterium) and lactobacillus (Lactobacillus) bacteria. According to any one of the above embodiments, the cell culture medium of the present invention can be any culture medium suitable for the growth of cells, such as natural culture medium or artificial culture medium such as balanced salt solution, basal culture medium or composite culture medium containing biological fluids. The cell culture medium of the present invention can be further supplemented as known in the art. According to the present invention, the cell culture medium is supplemented with ACC stabilized by at least one stabilizer. The stabilizer can be any agent known in the art. In a specific embodiment, the stabilizer is selected from polyphosphates such as inorganic polyphosphates, phosphorylated amino acids, bisphosphonates, organic acids and any combination thereof. According to some embodiments, the stabilizer is a combination of the stabilizers, such as a combination of an inorganic polyphosphate and an organic acid such as citric acid or a combination of a phosphorylated amino acid and an organic acid.

[0011] According to another aspect, the present invention provides amorphous calcium carbonate (ACC) stabilized by at least one stabilizer for use as a supplement to a cell culture medium. According to one embodiment, the stable ACC is added to the culture medium during culture medium preparation. According to another embodiment, the ACC is added to the culture medium before use.

[0012] According to yet another aspect, the present invention provides a cell culture medium supplement comprising amorphous calcium carbonate (ACC) stabilized by at least one stabilizer. According to one embodiment, the cell culture medium supplement comprising stable ACC is added to the culture medium during culture medium preparation. According to another embodiment, the supplement is added to the culture medium before use. The cell culture medium supplement can be a solid, liquid or semi-liquid supplement. According to any one of the above embodiments, ACC is stabilized by at least one stabilizer. Such a cell culture medium supplement can promote the growth of cells, tissues and organs, for example, promote the proliferation, maturation, reproduction, regeneration, development and / or differentiation of cells, tissues and organs.

[0013] According to another aspect, the present invention provides amorphous calcium carbonate (ACC) stabilized by at least one stabilizer and formulated as a cell culture medium supplement, wherein the ACC is stabilized by at least one stabilizer.

[0014] According to another aspect, the present invention provides a method for promoting cell growth of a biological culture, comprising exposing the biological culture to ACC stabilized by at least one stabilizer. According to some embodiments, the biological culture is selected from a culture of cells, a cell culture, a tissue culture, an organ culture, and a bacterial culture. In one embodiment, the method comprises promoting cell growth, and in particular promoting the formation of myotubes, promoting embryonic development, promoting neural cell regeneration, and promoting the maturation and / or preservation of gametes.

[0015] According to one aspect, the present invention provides a kit comprising: ACC, the ACC is used as a supplement for the cell culture medium of the present invention; or a cell culture medium supplement comprising stable ACC; and instructions for use of the ACC or the supplement in combination with a cell culture medium. According to certain aspects, the present invention provides a kit comprising amorphous calcium carbonate (ACC) stabilized by at least one stabilizer and instructions for use of the ACC in combination with a cell culture medium. According to one embodiment, ACC is used as a cell culture medium supplement. According to some embodiments, the kit includes a cell culture medium supplement containing ACC stabilized by at least one stabilizer and instructions for use of the ACC in combination with a cell culture medium. The kit of the present invention may also include any known culture medium suitable for the growth of cells, tissues or organs.

[0016] This application also provides the following items:

[0017] 1. A cell culture medium, wherein the cell culture medium is supplemented with amorphous calcium carbonate (ACC) stabilized by at least one stabilizer, and wherein the cell culture medium is suitable for growing a biological culture.

[0018] 2. The cell culture medium of item 1, wherein the growth of the biological culture is selected from at least one of proliferation, maturation, reproduction, regeneration, cryopreservation or differentiation of the biological culture.

[0019] 3. The cell culture medium of item 1 or 2, wherein the cell culture medium is capable of promoting the growth of the biological culture.

[0020] 4. The cell culture medium according to any one of items 1 to 3, wherein the biological culture is selected from a culture of eukaryotic cells or prokaryotic cells.

[0021] 5. The cell culture medium according to item 4, wherein the culture of the eukaryotic cells is selected from the group consisting of culture of animal cells, plant cells and insect cells.

[0022] 6. The cell culture medium of item 5, wherein the culture of the animal cells is selected from the group consisting of cell culture, tissue culture, organ culture, stem cells, gametes, embryos and organs of humans or non-human mammals.

[0023] 7. The cell culture medium of item 6, wherein the mammalian cell culture is selected from the group consisting of neural, muscle, epithelial, bone, fat, stem cell and blood cell cultures.

[0024] 8. The cell culture medium according to any one of items 5 to 7, wherein the culture of cells is a primary cell culture or a cell line selected from a finite cell line and a continuous cell line.

[0025] 9. The cell culture medium of item 8, wherein the cell line is selected from FM3, HeLa, 293, A-549, ALC, CHO, HB54, HL60, COS-7, HEK293, VERO, BHK, CV1, MDCK, 3T3, C127, MRC-5, BAE-1, SH-SY5Y, L-929, HEP G2, NSO, U937, NAMALWA, WEHI 231, YAC 1 and U 266B1 cell lines.

[0026] 10. The cell culture medium of item 6, wherein the mammalian tissue culture is selected from the group consisting of epithelial tissue culture, connective tissue culture, muscle tissue culture and neural tissue culture.

[0027] 11. The cell culture medium of item 10, wherein the epithelial tissue culture is selected from the group consisting of skin, stomach and intestinal lining, kidney and glandular tissue culture, the muscle tissue culture is selected from the group consisting of smooth muscle, skeletal muscle and cardiac muscle tissue culture, and the neural tissue culture is selected from the group consisting of brain, spinal cord and neural tissue.

[0028] 12. The cell culture medium of item 6, wherein the stem cells are selected from embryonic stem cells, chorionic stem cells, amniotic membrane stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, NOM stem cells, glial stem cells, adult stem cells and induced pluripotent stem cells.

[0029] 13. The cell culture medium according to item 6, wherein the embryo is a human embryo or a non-human mammal embryo selected from the group consisting of livestock animal, pet and rodent embryos.

[0030] 14. The cell culture medium according to item 6, wherein the organ culture or the organ is an ovary, and / or the gamete cells are selected from oocytes or sperm cells.

[0031] 15. The cell culture medium of item 5, wherein the culture of the plant cells is selected from the group consisting of plant cells and tissue cultures; and the culture of the insect cells is selected from the group consisting of insect cells, tissues and organ cultures.

[0032] 16. The cell culture medium according to any one of items 5 and 15, wherein the culture medium is a natural culture medium selected from biological fluids, tissue extracts and clots; or an artificial culture medium selected from balanced salt solutions, basal culture media and complex culture media.

[0033] 17. The cell culture medium according to item 16, wherein the culture medium is a serum-free culture medium.

[0034] 18. The cell culture medium of item 16 or 17, wherein the cell culture medium is selected from DMEM / F-12, cleavage medium, DMEM, RPMI 1640, MEM, IMDM, L-15 medium (Leibovitz), MCDB medium, medium 199, opti-MEM, Schneider Drosophila medium, Grace insect medium, IPL-41 insect medium Sf-900, serum-free insect medium, Shields and Sang M3 insect medium, TC-100 insect medium, TNM-FH insect medium, Ham's F-12, Ham's F-10, GMEM, Ames medium, Eagle's basic medium (BME), Claycomb, Click medium, Glasgow's minimum essential medium (GMEM), MegaCell medium, McCoy's 5A modified medium, NCTC medium, Williams medium E, Waymouth medium, TC-10IPL-10 medium, medium for sperm separation, washing or maturation, medium for fertilization, medium for embryo development, and medium for cryopreservation of embryos and / or gametes.

[0035] 19. The cell culture medium according to item 5, wherein the culture of the eukaryotic cells is a culture of yeast, and the cell culture medium is selected from the group consisting of YPD, YPG and YPAD.

[0036] 20. The cell culture medium of item 5, wherein the cell culture medium suitable for the growth of prokaryotes is selected from LB and M9, and optionally wherein the prokaryotes are selected from Bifidobacterium and Lactobacillus.

[0037] 21. The cell culture medium of any one of items 1 to 20, wherein the stabilizer is selected from the group consisting of polyphosphates, phosphorylated amino acids, organic acids, phosphorylated, phosphonated, sulfated or sulfonated organic compounds, phosphates or sulfates of hydroxycarboxylic acids, bisphosphonates, sugars and their derivatives, proteins, phosphorylated proteins, natural and synthetic biopolymers and their derivatives, and any combination thereof.

[0038] 22. The cell culture medium of item 21, wherein the stabilizer is selected from phosphoserine, triphosphate, adenosine triphosphate, adenosine diphosphate, phytic acid, citric acid, etidronic acid, pyrophosphate, ethanol, hexametaphosphate, chitin, and any combination thereof.

[0039] 23. The cell culture medium of any one of items 1 to 22, wherein the average diameter of the primary particles of the stable ACC is from about 10 nm to about 500 nm.

[0040] 24. A cell culture medium as described in claim 1, wherein the stabilizer is selected from polyphosphates, phosphorylated amino acids, bisphosphonates, organic acids and any combination thereof; and the cell culture medium is suitable for the growth of (i) muscle, nerve or bone cells or tissue culture; (ii) human or non-human mammalian embryos; (iii) stem cells; (iv) gametes; or (v) ovaries.

[0041] 25. The cell culture medium of item 24, wherein the cell culture medium is capable of promoting (i) neural cell regeneration; (ii) myocyte formation; (iii) stem cell differentiation; (iv) oocyte or sperm cell maturation; (v) embryo development or (vi) embryo or gamete cryopreservation.

[0042] 26. Amorphous calcium carbonate (ACC) stabilized by at least one stabilizer, for use as a supplement for cell culture media.

[0043] 27. Amorphous calcium carbonate (ACC) formulated as a supplement for a cell culture medium, wherein the ACC is stabilized by at least one stabilizer.

[0044] 28. A cell culture medium supplement comprising amorphous calcium carbonate (ACC) stabilized by at least one stabilizer.

[0045] 29. A kit comprising:

[0046] (i) The stable ACC as described in item 26 or 27;

[0047] (ii) a supplement as described in item 28; or

[0048] (iii) calcium chloride, sodium carbonate, at least one stabilizer and instructions for preparing stable ACC from said calcium chloride, sodium carbonate and at least one stabilizer; and

[0049] Instructions for using the stabilized ACC or the supplement in combination with a cell culture medium.

[0050] 30. The kit according to item 29, further comprising a cell culture medium.

[0051] 31. The ACC of item 26 or 27, the supplement of item 28, or the kit of item 29 or 30, wherein the cell culture medium is suitable for the growth of biological cultures.

[0052] 32. The ACC, supplement or kit of claim 31, wherein the growth of the biological culture is selected from at least one of proliferation, maturation, reproduction, regeneration, development, cryopreservation and differentiation of the biological culture.

[0053] 33. The ACC, supplement or kit of claim 31 or 32, wherein the ACC is capable of promoting the growth of the biological culture.

[0054] 34. The ACC, supplement or kit of any one of items 31 to 33, wherein the biological culture is selected from a culture of eukaryotic cells or prokaryotic cells.

[0055] 35. The ACC, supplement or kit of claim 34, wherein the culture of eukaryotic cells is selected from the group consisting of animal cells, plant cells, insect cells and yeast cultures.

[0056] 36. The ACC, supplement or kit of claim 35, wherein the culture of animal cells is selected from cell culture, tissue culture, organ culture, stem cells, embryos, gametes and organs of humans or non-human mammals; the culture of plant cells is selected from plant cell culture and plant tissue culture; and the culture of insect cells is selected from insect cell, tissue and organ culture.

[0057] 37. The ACC, supplement or kit of claim 36, wherein (i) the mammalian cell culture is selected from the group consisting of neural, muscle, epithelial, bone, fat, stem cell and blood cell cultures; (ii) the mammalian tissue culture is selected from the group consisting of epithelial tissue culture, connective tissue culture, muscle tissue culture and neural tissue culture; (iii) the stem cell is selected from the group consisting of embryonic stem cells, chorionic stem cells, amniotic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, glial stem cells, adult stem cells and induced pluripotent stem cells; (iv) the embryo is a human embryo or a non-human mammalian embryo selected from the group consisting of livestock, pets and rodent embryos; (v) the gamete is selected from the group consisting of an oocyte and a sperm cell; or (vi) the organ culture or the organ is an ovary.

[0058] 38. The ACC, supplement or kit of claim 36 or 37, wherein (i) the culture of cells is a primary cell culture or a cell line selected from a finite cell line and a continuous cell line, optionally wherein the cell line is selected from the group consisting of FM3, HeLa, 293, A-549, ALC, CHO, HB54, HL60, COS-7, HEK293, VERO, BHK, CV1, MDCK, 3T3, C127, MRC-5, BAE-1, SH-SY5Y, L-929, HEP G2, NSO, U937, NAMALWA, WEHI 231, YAC 1 and U 266B1 cell lines; or (ii) the epithelial tissue culture is selected from the group consisting of skin, stomach and intestinal lining, kidney and glandular tissue culture, the muscle tissue culture is selected from the group consisting of smooth muscle, skeletal muscle and cardiac muscle tissue culture, and the neural tissue culture is selected from the group consisting of brain, spinal cord and neural tissue.

[0059] 39. An ACC, supplement or kit as described in any one of items 26 to 38, wherein the cell culture medium is selected from (i) a natural culture medium selected from a biological fluid, a tissue extract and a clot; (ii) an artificial culture medium selected from a balanced salt solution, a basal culture medium and a complex culture medium, wherein the artificial culture medium is optionally a serum-free culture medium; (iii) a culture medium suitable for the growth of prokaryotes selected from YT and M9; and (iv) a culture medium suitable for the growth of yeast selected from YPD, YPG and YPAD.

[0060] 40. The ACC, supplement or kit of item 39, wherein the cell culture medium is selected from DMEM / F-12, cleavage medium, DMEM, RPMI 1640, MEM, IMDM, L-15 medium (Leibovitz), MCDB medium, medium 199, opti-MEM, Schneider Drosophila medium, Grace insect medium, IPL-41 insect medium Sf-900, serum-free insect medium, Shields and Sang medium. M3 insect medium, TC-100 insect medium, TNM-FH insect medium, Ham's F-12, Ham's F-10, GMEM, Ames medium, Eagle's basic medium (BME), Claycomb, Click medium, Glasgow's minimum essential medium (GMEM), MegaCell medium, McCoy's 5A modified medium, NCTC medium, Williams medium E, Waymouth medium, TC-10IPL-10 medium, medium for sperm separation, washing or maturation, medium for fertilization, medium for embryo development, and medium for cryopreservation of embryos or / and gametes.

[0061] 41. The ACC, supplement or kit of any one of items 26 to 40, wherein the stabilizer is selected from the group consisting of polyphosphates, phosphorylated amino acids, organic acids, phosphorylated, phosphonated, sulfated or sulfonated organic compounds, phosphates or sulfates of hydroxycarboxylic acids, bisphosphonates, carbohydrates, their derivatives, proteins, phosphorylated proteins, natural and synthetic biopolymers and their derivatives, and any combination thereof.

[0062] 42. The ACC, supplement or kit of any one of items 26 to 41, wherein the average diameter of the primary particles of the stable ACC is from about 10 nm to about 500 nm.

[0063] 43. A method for promoting the growth of a biological culture comprising exposing the biological culture to ACC stabilized by at least one stabilizer.

[0064] 44. A method as described in item 43, wherein promoting the growth of the biological culture is selected from at least one of promoting proliferation, maturation, reproduction, regeneration, differentiation or development of the biological culture.

[0065] 45. The method of claim 44, wherein the biological culture is selected from mammalian cell culture, tissue culture and organ culture.

[0066] 46. ​​A method as described in any of items 43 to 45, wherein the cell culture is selected from neural, muscle, epithelial, bone, fat, stem cell, gamete cell and blood cell cultures; the mammalian tissue culture is selected from epithelial tissue culture, connective tissue culture, muscle tissue culture and neural tissue culture; and the organ culture is selected from ovary.

[0067] 47. The method of item 46, comprising promoting the formation of myotubes.

[0068] 48. The method of item 47, further comprising reducing the onset of contractile activity in early developing myotubes.

[0069] 49. The method of item 47 or 48, wherein the muscle cells formed from the myotubes are selected from skeletal muscle cells and cardiac muscle cells.

[0070] 50. The method of claim 46, comprising accelerating nerve cell regeneration.

[0071] 51. The method of claim 46, comprising promoting embryonic development in vitro.

[0072] 52. The method of claim 51, wherein promoting embryo development comprises increasing the proportion of embryos that reach a developmental stage and / or the time to reach a developmental stage, wherein the developmental stage is selected from the group consisting of compaction, blastocyst formation, and blastocyst hatching stages.

[0073] 53. A method as described in item 51 or 52, wherein the embryo is a human or non-human mammal embryo.

[0074] 54. The method of claim 53, wherein the non-human mammal embryo is selected from the group consisting of livestock animal, domestic pet, rodent and primate embryos.

[0075] 55. The method of item 46, comprising accelerating stem cell differentiation and / or proliferation.

[0076] 56. The method of item 46, comprising promoting the maturation and / or preservation of gametes selected from oocytes and sperm cells.

[0077] 57. The method of item 56, wherein promoting the maturation of sperm cells comprises promoting sperm motility, promoting sperm progressive motility, increasing sperm count, and any combination thereof.

[0078] 58. The method of any one of items 43 to 57, wherein exposing the biological culture to ACC stabilized by at least one stabilizer comprises adding the ACC to a cell culture medium.

[0079] 59. A method as described in any of items 43 to 58, wherein the stabilizer is selected from the group consisting of: polyphosphates, phosphorylated amino acids, organic acids, phosphorylated, phosphonated, sulfated or sulfonated organic compounds, phosphates or sulfates of hydroxycarboxylic acids, bisphosphonates, carbohydrates and their derivatives, proteins, peptides, phosphorylated proteins or peptides, natural and synthetic biopolymers and their derivatives, and any combination thereof.

[0080] 60. A method as described in item 59, wherein the stabilizer is selected from phosphoserine, triphosphate, adenosine triphosphate, adenosine diphosphate, phytic acid, citric acid, etidronic acid, pyrophosphate, ethanol, hexametaphosphate, chitin and any combination thereof.

[0081] 61. The method of any one of items 43 to 60, wherein the average diameter of the primary particles of the stable ACC is from about 10 nm to about 500 nm. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1The effects of different calcium sources on neuronal sprouting from cultured spinal cord dorsal root ganglia (SC-DRG) slices are shown. 2+ Immunofluorescence staining (anti-neurofilament antibody) of nerve fibers grown from SC-DRG slices with (A) ACC-etidronic acid; (B) ACC-phosphoserine; (C) gastrolith; (D) crystalline calcium carbonate (CCC); and (E) CaCl2 solution (control). Original magnification x100.

[0084] Figure 2 The effects of (A) ACC stabilized by etidronic acid and (B) CaCl2 solution (control) on neuronal sprouting from brain cells cultured on chitosan microcarriers (MC) are shown. Immunofluorescence staining of neurofilaments (anti-neurofilament antibody) grown from brain cell-chitosan MC aggregates after 30 days of culture in the presence of 2 mM ACC-etidronic acid or CaCl2 is presented.

[0085] Figure 3 Shown is the effect of ACC on myotube formation in healthy skeletal muscle cultures. Original magnification x40. Skeletal muscle cultures were exposed to the following calcium compounds (final Ca 2+ Concentration 2mM): ACC-etidronic acid; ACC-ADP; gastric stone; crystalline calcium carbonate (CCC); and CaCl2 solution (control). Cultures were fixed after 4 and 7 days and stained with Giemsa. Promotion of myotube formation from skeletal muscle cultures was observed in cells treated with ACC.

[0086] Figure 4 Shown is the effect of ACC in culture medium on early myotube formation in mdx cell line cultures. Shown is Giemsa staining of cultures exposed to medium containing CaCl2, ACC-ET and ACC-phosphoserine (ACC-PS). Original magnification X100.

[0087] Figure 5 Shown are creatinine kinase (CK) levels as measured in mdx myocyte cell lines exposed to two ACC preparations (ACC-ET and ACC-PS) and CaCl2.

[0088] Figure 6 Shown are the effects of ACC (ACC-PS, ACC-PP, and control (CaCl2)) on myotube formation in primary cultures of mdx mice (Giemsa staining; original magnification X50).

[0089] Figure 7Shown are the effects of ACC on myotube formation in primary cultures of mdx mice as demonstrated by myosin immunostaining; control (CaCl2); ACC-PS, ACC-polyphosphate (ACC-PP). Original magnification X100.

[0090] Figure 8 Shown is the effect of stabilized ACC on the in vitro development of mouse embryos.

[0091] Figures 9A-9C Figure 2 shows the changes of Alizarin red staining of osteoblasts after 10 days of culture with various medium treatments. The medium was supplemented with additional 1 mM Ca 2+ , which come from: A-ACC, B-CaCl2, or C-control, no Ca 2+ Add to.

[0092] Figures 10A-10C Figure 2 shows the changes in alkaline phosphatase staining of osteoblasts after 10 days of culture with various culture medium treatments. Culture medium c was supplemented with an additional 1 mM Ca 2+ , which come from: A-ACC, B-CaCl2, or C-control, no Ca 2+ Add to.

[0093] Figures 11A-11F The results show that the addition of 1 mM Ca from different sources 2+ Alizarin red (AC) and alkaline phosphatase (DF) staining of mdx cell lines grown in medium containing: A and D - ACC, B and E - CaCl2, or C and F - control (no additional calcium supplement).

[0094] Figures 12A-12B Shown are the effects of stabilized ACC in in vitro cultured ovaries (A), where granulosa cells surrounding oocytes are intact, and controls (B) (no ACC in culture medium), where intact granulosa cells and oocytes at the germinal vesicle stage were not observed. DETAILED DESCRIPTION OF THE INVENTION

[0096] Surprisingly, it was found that adding ACC to a cell culture medium promotes the growth of various types of cells. According to one aspect, the present invention provides a cell culture medium supplemented with amorphous calcium carbonate (ACC), wherein the ACC is stabilized by at least one stabilizer. According to other aspects, the present invention provides stable ACC for use as a supplement to a cell culture medium. In certain aspects, the present invention provides a cell culture supplement comprising stable ACC.

[0097] For each aspect of the present invention, the following terms are used individually and collectively, and the specific parameters are defined as follows:

[0098] The terms "cell culture medium", "growth medium" and "culture medium" are used interchangeably herein and refer to a culture medium used for, suitable for or capable of supporting the growth of biological cultures such as cells, tissues or organs and providing a suitable environment for the cells, tissues or organs. Different cell culture media may have different properties and contain different components, however almost all culture media are isotonic culture media and have an osmotic pressure suitable for cell growth. Therefore, a cell culture medium is an isotonic cell culture medium. The term "isotonic" as used herein refers to a cell culture medium having an osmotic pressure of an aqueous solution in the range of 270-300mOsmol / kg at 37°C. Therefore, in one embodiment, water or more specifically deionized water itself is not considered a cell culture medium. According to some embodiments, the tissue culture comprises sodium chloride, potassium chloride and a phosphorus source, such as sodium phosphate monobasic or dibasic. According to some embodiments, the culture medium is suitable for growing cell cultures, tissue cultures, organ cultures or organs. The cells may be eukaryotic cells or prokaryotic cells. Specifically, a cell culture medium refers to a culture medium suitable for the growth of eukaryotic cell cultures, tissue cultures or organ cultures. In some specific embodiments, the culture medium can be a complete culture medium, a basal culture medium, a basal culture medium supplemented with a cell culture medium supplement, a culture medium with various amounts of serum, or a chemically defined culture medium.

[0099] The term "supplement" as used herein refers to a culture medium to which ACC stabilized with at least one stabilizer has been added, so that the culture medium comprises ACC stabilized with at least one stabilizer. The term encompasses a culture medium prepared with ACC and a culture medium to which ACC is added prior to use.

[0100] Thus, in one embodiment, the present invention provides a cell culture medium, wherein the cell culture medium is supplemented with amorphous calcium carbonate (ACC) stabilized by at least one stabilizer, and wherein the cell culture medium is suitable for the growth of a culture of an organism.

[0101] The terms "culture", "biological culture" and "culture of cells" are used interchangeably herein and refer to cells, tissues, organ cultures or organs grown in vitro under predefined conditions. In some embodiments, the biological culture is selected from animal, plant or insect cell culture; animal, plant or insect tissue culture; animal, plant or insect organ culture, yeast culture and bacterial culture.

[0102] The term "cell culture" as used herein refers to cells of multicellular eukaryotic organisms maintained, cultured or grown in an artificial in vitro environment. Cell culture can be a suspension culture, in which cells are cultured in a liquid medium by continuous agitation or cultured on microcarriers or in adherent or monolayer cultures.

[0103] As used herein, the term "tissue culture" refers to tissue maintained or grown in vitro.

[0104] As used herein, the term "organ culture" refers to a part or a whole organ cultured in vitro.

[0105] The term "stem cell" refers to cells that have the ability to proliferate and differentiate into different cell types.

[0106] The terms "amorphous calcium carbonate" and "ACC" are used interchangeably herein and refer to a non-crystalline form of calcium carbonate stabilized by at least one stabilizer. ACC can be obtained from natural sources or chemically synthesized. The term also includes naturally stabilized ACC, such as ACC obtained from gastric stones.

[0107] As used herein, the term "natural ACC" refers to any ACC isolated or derived from a natural source. Non-limiting examples of natural sources of ACC include gastroliths of freshwater crustaceans.

[0108] As used herein, the term "synthetic ACC" refers to any ACC produced and / or derived from humans ex vivo.

[0109] According to some embodiments, the cell culture medium according to the present invention is suitable for the growth of biological cultures. According to one embodiment, the biological culture is a culture of eukaryotic or prokaryotic cells. As used herein, the term "growth" encompasses any of the following: proliferation, maturation, reproduction, regeneration, support, differentiation, development, preservation, cryopreservation, and any combination thereof, and can be used differently depending on the cell type. According to one embodiment, the cell culture medium is suitable for the support, proliferation, or reproduction of cells. In an exemplary embodiment, the cell culture medium is suitable for the proliferation or reproduction of eukaryotic cells, such as unicellular organisms or multicellular organisms. According to another exemplary embodiment, the cell culture medium is suitable for the proliferation or reproduction of prokaryotic cells. According to other embodiments, the cell culture medium is suitable for the maturation and / or development of cells, such as the development of embryos or the maturation of gamete cells. As used herein, the term "embryo" refers to the fertilized oocyte of a mammal, i.e., a zygote, and refers to a multicellular organism at its earliest developmental stage developed from the zygote. The terms "gamete" or "gamete cell" are used interchangeably herein and refer to any male or female reproductive cell capable of initiating the formation of a new diploid individual. Examples of gametes are sperm and oocytes. As used herein, the terms "sperm" and "spermatozoa" are used interchangeably and refer to male reproductive cells. The term "sperm sample" refers to one or more samples containing sperm. The sperm sample can be semen obtained from a subject or processed semen, liquefied semen, precipitated and optionally resuspended sperm, etc. According to some embodiments, the cell culture medium is suitable for the regeneration of cells, such as the regeneration of nerve cells. As used herein, the terms "neuronal regeneration" and "neuroregeneration" can be used interchangeably and refer to the recovery of the function of damaged nerves. Specifically, it includes the recovery of signal transmission through nerves, and the recovery is by repairing the regrowth of damaged parts, peripheral or central nervous system axons and dendrites neuronal fibers. In some embodiments, neuroregeneration refers to sprouting from damaged neuronal fibers. In some embodiments, neuroregeneration refers to sprouting from damaged neuronal fibers. Therefore, the cell culture medium according to the present invention can promote the regeneration of deep (profound) nerve fibers. According to another embodiment, the cell culture medium is suitable for the differentiation of cells, such as the differentiation of stem cells, and in particular the differentiation of stem cells to osteoblasts. According to some embodiments, the cell culture medium is suitable for supporting the proliferation of tissue cultures and organ cultures. According to some embodiments, the cell culture medium is suitable for in vitro preservation of the organ.

[0110] According to some embodiments, the cell culture medium supplemented with stable ACC according to the present invention can promote the growth as defined above of biological culture. As used herein, the term "promote" refers to promoting (promoting), improving (improving), enhancing (augmenting), improving (ameliorating), typically increasing growth parameters. According to some embodiments of the present invention, the terms "can promote" and "promote" are used interchangeably. Promotion can be measured relative to a control sample grown in the same conditions but without ACC. Therefore, in one embodiment, the cell culture medium supplemented with stable ACC according to the present invention can promote the proliferation, maturation, reproduction, regeneration, development, cryopreservation and / or differentiation of cells, tissues or organs. According to one embodiment, the cell culture medium can promote the differentiation of stem cells. According to another embodiment, the cell culture medium supplemented with stable ACC can promote the proliferation of cells. According to another embodiment, the cell culture medium supplemented with stable ACC can promote the maturation of cells. According to another embodiment, the cell culture medium supplemented with stable ACC can promote the development of cells or tissues. According to certain embodiments, the cell culture medium supplemented with stable ACC can promote cell regeneration. The promotion of proliferation, maturation, reproduction, regeneration, development and / or differentiation of cells, tissues or organs can be measured as the promotion percentage compared with the control defined above. Therefore, according to one embodiment, the cell culture medium supplemented with stable ACC can increase growth parameters by about 10% to about 600%, about 20% to about 500%, about 30% to about 400%, about 40% to about 300%, about 50% to about 200%, about 60% to about 150% or about 70% to about 100%. Increasing by 100% means that parameters (such as proliferation) are increased to 2 times; Increasing by 200% means that parameters (such as embryonic development) are increased to 3 times, etc. According to some embodiments, growth parameters are increased by about 100% to about 500%, about 120% to about 400%, about 150% to about 300%.

[0111] According to some embodiments, the cell culture medium supplemented with stable ACC is suitable for eukaryotic cell culture, such as the growth of eukaryotic cells, tissue or organ cultures and optionally promotes their growth. According to some embodiments, the cell culture medium supplemented with stable ACC is used for the growth of eukaryotic cells. According to some embodiments, the culture of eukaryotic cells is selected from the culture of animal, plant and insect cells.

[0112] According to one embodiment, the culture of eukaryotic cells, tissue or organ cultures are animal cells, tissue or organ cultures, stem cells, embryos and organs. According to some embodiments, the animal is a human or non-human mammal. Therefore, according to one embodiment, the cell culture medium supplemented with stable ACC is suitable for the growth of mammalian cells, tissue or organ cultures, mammalian stem cells, mammalian embryos or mammalian organs and optionally promotes their growth.

[0113] According to one embodiment, the mammal is a human, thus according to one embodiment the cell culture medium according to the invention is suitable for the culture of human cells, tissue or organ cultures, stem cells, embryos or the growth of organs.

[0114] According to another embodiment, the mammal is a non-human mammal. According to one embodiment, the non-human mammal is a livestock animal, such as a cow, pig, sheep, goat, horse, mule, donkey, buffalo, or camel. In another embodiment, the non-human mammal is a domestic pet, such as a cat or dog; a rodent, such as a mouse, rat, guinea pig, or hamster; a lagomorph, such as a rabbit; or a primate, such as a monkey (e.g., a macaque) or an ape (e.g., a chimpanzee).

[0115] According to some embodiments, the cell culture medium according to the present invention is suitable for the growth of mammalian cell cultures. According to some embodiments, the mammalian cell culture, human or non-human mammalian cell culture is selected from the cell culture of nerves, muscles, epithelium, bone, fat, stem cells, gametes and blood cells. According to one embodiment, the cell culture is a muscle cell culture. According to another embodiment, the cell culture is a neural cell culture. According to another embodiment, the cell culture is a cell culture of bone or osteocytes. According to one embodiment, the cell culture is a bone marrow cell culture. According to another embodiment, the cell culture is a tumor or cancer cell. According to some embodiments, the cell culture medium supplemented with ACC according to the present invention is capable of promoting the growth of the cell culture. According to some embodiments, the cell culture is a suspension or adherent cell culture.

[0116] According to some embodiments, the cell culture is a primary culture.The term "primary culture" as used herein refers to cells isolated from a tissue and proliferated under appropriate conditions.

[0117] According to another embodiment, the cell culture is a cell line. The terms "subculture" and "cell line" are used interchangeably herein and refer to a subcultured primary culture, i.e., a primary culture that is transferred from one culture vessel to another culture vessel. According to some embodiments, the cell line is a finite cell line, i.e., a cell line that has a finite lifespan and undergoes a finite number of cell generations. According to another embodiment, the cell line is a continuous cell line, i.e., an immortal cell line that has acquired the ability to divide indefinitely.

[0118] According to a specific embodiment, the cell line is selected from FM3, HeLa, 293, A-549, ALC, CHO, HB54, HL60, COS-7, HEK293, VERO, BHK, CV1, MDCK, 3T3, C127, MRC-5, BAE-1, SH-SY5Y, L-929, HEPG2, NSO, U937, NAMALWA, WEHI 231, YAC 1 and U 266B1 cell lines. According to some embodiments, the cell culture medium supplemented with stable ACC can promote the growth of the cell line.

[0119] According to some embodiments, the cell culture medium of the present invention is suitable for the growth of mammalian tissue cultures. According to one embodiment, the mammalian tissue culture is a human tissue culture. According to another embodiment, the tissue culture is a non-human mammalian tissue culture. According to one embodiment, the tissue culture is selected from epithelial tissue, connective tissue, muscle tissue and neural tissue culture. According to one embodiment, the tissue culture is a neural tissue culture. According to another embodiment, the tissue culture is a muscle tissue culture. According to yet another embodiment, the tissue culture is an epithelial tissue culture, such as the nasal olfactory mucosa. According to another embodiment, the tissue culture is a bone tissue culture.

[0120] According to some more specific embodiments, the tissue culture is selected from kidney, liver, glandular, brain, bone, eye and muscle tissue cultures. According to one embodiment, the epithelial tissue culture is selected from skin, stomach and intestinal lining, kidney and glandular tissue cultures, the muscle tissue culture is selected from smooth muscle, skeletal muscle and cardiac muscle tissue cultures, and the neural tissue culture is selected from brain, spinal cord and neural tissue.

[0121] According to some embodiments, the cell culture medium supplemented with stable ACC can promote the growth of the tissue culture. In one embodiment, such a culture medium can promote the regeneration of neural tissue culture. In some embodiments, the culture medium can promote the regeneration of neural culture by about 10% to about 200%, about 20% to about 150%, about 30% to about 120% or about 40% to about 100%. In other embodiments, such a culture medium can promote the formation of myotubes and / or promote or promote the contractility of muscle cells, for example, also reduce the time when the spontaneous contraction activity of myotubes begins. In one embodiment, the culture medium can promote the formation of myotubes by about 10% to about 600%, about 20% to about 500%, about 30% to about 400%, about 40% to about 300%, about 50% to about 200%, about 60% to about 150% or about 70% to about 100%. As used herein, the term "myotube formation" refers to the process in which myoblasts fuse into multinuclear fibers-myotubes.

[0122] According to some embodiments, the cell culture medium of the present invention is suitable for stem cell growth. According to some embodiments, the stem cell is a human stem cell. According to another embodiment, the stem cell is a non-human mammalian stem cell. According to some embodiments, the stem cell is selected from embryonic stem cells, chorionic stem cells, amniotic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, glial stem cells, adult stem cells and induced pluripotent stem cells. According to one embodiment, the stem cell is an embryonic stem cell. According to another embodiment, the stem cell is a hematopoietic stem cell. According to another embodiment, the stem cell is a mesenchymal stem cell. According to yet another embodiment, the stem cell is a pluripotent stem cell. According to some embodiments, the stem cell is an adult stem cell, such as a mesenchymal stem cell, an epidermal stem cell, an epithelial stem cell, a hematopoietic stem cell or a neural stem cell. According to one embodiment, the cell culture medium of the present invention is suitable for the proliferation, expansion and / or differentiation of stem cells. According to another embodiment, the cell culture medium of the present invention can promote the proliferation and / or differentiation of stem cells. According to a specific embodiment, the cell culture medium of the present invention can promote the differentiation of stem cells into osteoblasts, i.e., promote osteoblast differentiation.

[0123] According to some embodiments, the cell culture medium of the present invention is suitable for the growth of embryos. According to one embodiment, the cell culture medium of the present invention is suitable for the development of embryos. According to another embodiment, the cell culture medium of the present invention can promote the development of embryos. According to one embodiment, the culture medium can promote the development of embryos by about 10% to about 300%, about 20% to about 250%, about 30% to about 200%, about 40% to about 150%, about 60% to about 100% or about 70% to about 90%. The terms "embryogenesis" and "embryo development" are used interchangeably herein and refer to the process that embryos are formed and developed from the zygote stage to become embryos, as known in the art, and include the stage of reaching cleavage, compaction, blastocyst formation or blastocyst hatching stage. The terms "cleavage", "compaction", "blastocyst" and "hatching" used herein refer to the terms conventionally used in embryology. The term "cleavage" refers to the division of cells in early embryos. Produce a cell cluster identical in size to the original zygote. The different cells derived from cleavage are called blastomeres (blastomere). As used herein, the term "compaction" refers to a stage in which the cleaved cells from the zygote maximize their contact with each other by polarization and adhesion, forming a compact ball that is held together by tight connection. As used herein, the term "blastocyst" refers to the structure formed after the compaction stage, and comprises the inner cell mass that forms the embryo subsequently and the blastocyst outer layer that surrounds the inner cell mass and the fluid-filled cavity called the blastocyst cavity (blastocoele). As used herein, the term "hatching" refers to the stage in which the embryo appears by its shell (zona pellucida (zona pellucida)).

[0124] As used herein, the term "promoting embryonic development" refers to the ratio of the embryo that promotes, promotes or improves the rate and / or effectiveness of the developmental process and the successful growth and development. Promoting is measured by a control sample relative to the same program of experience but without ACC in the growth medium. According to one embodiment, the embryo is a human embryo. According to another embodiment, the embryo is a non-human mammal embryo. In some embodiments, the non-human mammal is selected from cattle, pigs, sheep, goats, horses, mules, donkeys, buffaloes or camels. In some other embodiments, the non-human mammal is selected from cats, dogs, mice, rats, guinea pigs, hamsters, rabbits, monkeys or apes.

[0125] According to some embodiments, the cell culture medium is capable of improving or prolonging cryopreservation of embryos or gametes, as described above. As used herein, the term "cryopreservation" refers to the storage of cells, such as gametes of embryos, at ultra-low temperatures, typically in liquid nitrogen (-196°C).

[0126] According to some embodiments, the cell culture medium of the present invention is suitable for gamete cell growth. According to one embodiment, the cell culture medium supplemented with stable ACC is suitable for the maturation and / or preservation of gamete cells. According to one embodiment, the gamete cell is an oocyte. According to another embodiment, the gamete cell is a sperm cell. According to any one of the above embodiments, the gamete cell is a gamete cell of a human or non-human mammal. According to some embodiments, the non-human mammal is selected from the group consisting of livestock animals, domestic pets, rodents, wild animals and primates. In one embodiment, livestock animals are selected from cattle, pigs, sheep, goats, horses, mules, donkeys, buffaloes and camels. In some other embodiments, domestic pets are cats or dogs, rodents are rats, mice, guinea pigs or hamsters, lagomorphs are rabbits, and primates are monkeys such as macaques or apes such as chimpanzees.

[0127] According to another embodiment, the gamete cell is a non-mammalian gamete cell. According to some embodiments, the non-mammal is selected from the group consisting of fish, insects and birds.

[0128] According to one embodiment, the gamete cell is a human sperm cell or an oocyte.

[0129] According to some embodiments, a cell culture medium supplemented with stable ACC can promote the maturation of oocytes or sperm. According to one embodiment, a cell culture medium supplemented with stable ACC can improve sperm quality. The terms "promoting sperm maturation" and "improving sperm quality" are used interchangeably herein and refer to improving sperm quality, such as promoting sperm motility, promoting sperm forward motility, increasing sperm counts, and any combination thereof. Therefore, in one embodiment, a cell culture medium supplemented with stable ACC can promote sperm motility, promote sperm forward motility, increase sperm counts, and any combination thereof. The term "sperm motility" as used herein refers to the proportion of sperm moving in all sperm of a given sample sample. The term "forward motility" as used herein refers to the proportion of sperm moving in a roughly constant direction. The terms "promoting sperm motility" and "promoting sperm forward motility" as used herein refer to the proportion of sperm that increase movement and sperm with forward motility, respectively. Therefore, in one embodiment, the present invention provides a method for promoting sperm motility. According to another aspect, the present invention provides a method for promoting sperm forward motility. Sperm motility, forward motility and sperm maturation stage can be assessed by any method known in the art. For example, motility can be assessed by computer-assisted sperm analysis (CASA) method (Amann & Waberski, 2014, Theriogenology, 81: 5-17). The term "sperm sample" refers to one or more samples containing sperm. The sperm sample can be semen obtained from a subject or processed semen, liquefied semen, precipitated and optionally resuspended sperm, etc. According to some embodiments, increasing sperm counts includes increasing sperm counts in a motility or forward motility program. According to one embodiment, the motility or forward motility program is an upstream program.

[0130] According to some embodiments, the cell culture medium of the present invention is suitable for the growth of an organ tissue or organ. According to some embodiments, the organ tissue or organ is selected from the group consisting of ovary, cornea, heart, kidney, pancreas, liver, spleen, lung, testis, bladder and blood vesicle. In a specific embodiment, the organ tissue or organ is ovary. Therefore, the cell culture medium of the present invention is capable of promoting the preservation or maintenance of an organ tissue or organ.

[0131] According to some embodiments, the cell culture medium of the present invention is suitable for the growth of plant cells. According to some embodiments, the cell culture medium of the present invention is suitable for the growth of plant cell cultures. According to another embodiment, the cell culture medium of the present invention is suitable for the growth of plant tissue cultures. The term "plant cell culture" refers to plant cells derived from plant tissue or cells, which are then cultured in a container or recipient. The term "plant tissue culture" includes callus tissue (callus), differentiated cultured tissue or cultured organ tissue. The term "callus" refers to a large number of unorganized parenchymatic cells derived from plant tissue (explant).

[0132] According to another embodiment, the cell culture medium of the present invention is suitable for the growth of insect cells. According to one embodiment, the insect cells are insect cell cultures, such as insect cell lines. The cell types are as described above. In a specific embodiment, the cell line is selected from Sf9, Sf21 and high-five cell lines. According to another embodiment, the cell culture medium of the present invention is suitable for the growth of insect tissue cultures. According to a further embodiment, the insect cells are insect organ cultures.

[0133] According to some embodiments, the cell culture is a suspension or adherent cell culture.

[0134] In one embodiment, the cell culture medium supplemented with stabilized ACC is capable of promoting the growth of plant cells or tissue cultures, or is capable of promoting the growth of insect cells, tissues or organ cultures.

[0135] According to any one of the above embodiments, the cell culture medium of the present invention, suitable for the growth of animal, plant or insect cells, tissue or organ cultures, can be a natural culture medium or an artificial culture medium, supplemented with ACC stabilized by at least one stabilizer, as defined in the present invention. According to some embodiments, the culture medium is a natural culture medium supplemented with ACC stabilized by at least one stabilizer. According to some embodiments, the natural culture medium comprises a biological fluid selected from plasma, serum, lymph, human placental cord serum and amniotic fluid. According to another embodiment, the natural culture medium comprises a tissue extract, such as an extract of liver, spleen, tumor, leukocytes and bone marrow, an extract of bovine embryo and chicken embryo. According to another embodiment, the natural culture medium comprises a coagulant or plasma clot. According to some embodiments, the culture medium is an artificial culture medium supplemented with ACC stabilized by at least one stabilizer. According to one embodiment, the artificial culture medium is a balanced salt solution. Examples of balanced salt solutions are PBS, DPBS, HBSS, EBSS Tyrode's T6, WM1, Pool's P1, Quinn's HTF and Gardner's G1. According to another embodiment, the artificial culture medium is a basal culture medium. According to some embodiments, the culture medium can be further supplemented as known in the art. According to one embodiment, the culture medium is supplemented with serum, such as fetal calf serum.According to a further embodiment, the artificial culture medium is a complex culture medium.

[0136] The term "basal medium" as used herein refers to a nutrient mixture of inorganic salts, sugars, amino acids, optionally also containing vitamins, organic acids and / or buffers. The basal medium and supplements together provide the nutrients necessary to support cell life, growth and reproduction. The choice of basal medium used should be adapted to the culture.

[0137] According to one embodiment, the artificial culture medium is a serum-free culture medium. According to a further embodiment, the artificial culture medium is a culture medium with reduced serum content. According to another embodiment, the artificial culture medium is a protein-free culture medium.

[0138] Examples of cell culture media that can be supplemented with ACC stabilized by at least one stabilizer and used according to the present invention are Dulbecco's modified Eagle medium (DMEM), minimal essential medium (EMEM), RPMI 1640 medium (developed at Roswell Park Memorial Institute) and Eagle basal medium (BME). Another example of a culture medium according to the present invention is Ham's nutrient mixture, such as Ham's F-10, Ham's F-12, DMEM / F-12 (DMEM and Ham's F-12). Other examples are Iscove's modified Dulbecco's medium (IMDM), opti-MEM and Glasgow's MEM (GMEM). Examples of culture media suitable for insect cell growth are IPL-41 insect medium, Schneider fruit fly medium, Grace insect medium, serum-free insect medium, Sf-900, TC-10, Shields and Sang M3 insect medium, TC-100 insect medium, TNM-FH insect medium and IPL-10. An example of a medium suitable for embryo growth is a monoculture medium such as SAGE 1-Step TM or continuous media such as Quinns Advantage TM Continuous medium (ORIGIO). Examples of culture media suitable for the growth of plant cells are Murashige and Skoog (MS), B5, N6 and Nitsch medium.

[0139] Other examples of culture media are Modified Medium, NCTC medium, MegaCell medium, Claycomb, Click medium, L-15 medium, Medium 199, MCDB medium, Ames medium, BGJb medium (Fitton-Jackson modified), Click medium, CMRL-1066 medium, McCoy's 5A modified medium, NCTC medium, Swim's S-77 medium, Waymouth medium, Willliam's medium E, and in vitro fertilization medium, e.g. GM501, SSM TM , cleavage K-SICM, blastocyst K-SIBM, Quinns Cleavage, Quinns blastocyst, FERTICULT TM IVF culture medium, FERTICULT TM G3 medium, IVC-TWO TM 、IVC-THREE TM , EmbryoAssist TM , BlastAssist TM 、ISM1、ISM2、G-1 TM PLUS, G-2 TM PLUS, IVF TM and CCM TM A further example of a culture medium is a culture medium for sperm isolation, e.g. PureCeption TM 、Multipurpose Handling Medium for sperm washing, such as Quinns TM Sperm washing medium, Multipurpose Handling or modified HTF medium with gentamicin; a medium for sperm capacitation, such as Biggers-Whitten-Whittingham (BWW) medium, Ham's-F10, and modified Tyrode's medium (HSM); and a medium for maturation, which enables immature oocytes to be cultured into fully developed embryos suitable for transfer. In other embodiments, the culture medium is a medium for oocyte maturation, such as SAGE TMIn vitro maturation medium (IVM) and BO-IVM oocyte maturation medium, medium for fertilization, medium for embryo development, medium for gamete processing, medium for preimplantation genetic diagnosis (PGD) or medium for embryo or / and gamete maturation, processing and / or cryopreservation. Thus, according to one embodiment, the cell culture medium is selected from the group consisting of DMEM, RPMI 1640, MEM, IMDM, L-15 medium (Leibovitz), MCDB medium, medium 199, opti-MEM and DMEM / F-12, Schneider's Drosophila medium, Grace's insect medium, IPL-41 insect medium Sf-900, serum-free insect medium, Shields and Sang M3 insect medium, TC-100 insect medium, TNM-FH insect medium, Ham's F-12, Ham's F-10, GMEM, Ames medium, Eagle's basic medium (BME), Claycomb, Click medium, Glasgow's minimum essential medium (GMEM), MegaCell medium, McCoy's 5A modified medium, NCTC medium, Williams medium E, Waymouth medium, TC-10 and IPL-10 medium. According to another embodiment, the cell culture medium is selected from DMEM, RPMI 1640, MEM, IMDM, opti-MEM, GMEM, Ham's F-12 and DMEM / F-12, Schneider's Drosophila medium, Grace's insect medium, Sf-900, TC-10, IPL-10 medium, medium for sperm isolation, washing or maturation, e.g. PureCeption TM 、MultipurposeHandling Quinns TM Sperm washing medium, Biggers-Whitten-Whittingham (BWW) medium, Ham's-F10 and modified Tyrode's medium (HSM) and modified HTF medium with gentamicin, medium for fertilization, medium for embryo development, and medium for embryo or / and gamete maturation, processing and / or cryopreservation.

[0140] According to some embodiments, the cell culture medium supplemented by the stable ACC of the present invention is suitable for the growth of unicellular eukaryotic organisms. According to one embodiment, the unicellular eukaryotic organism is a yeast (yeast), such as a yeast (Saccharomyce), more specifically a saccharomyces cerevisiae (Saccharomyces cerevisiae). According to one embodiment, the cell culture medium suitable for the growth of unicellular eukaryotic organisms (such as saccharomyces cerevisiae) of the present invention is selected from yeast extract peptone glucose (YPD), yeast extract peptone glycerol (YPG) and yeast extract peptone glucose (YPAD) culture medium. In one embodiment, the cell culture medium supplemented with stable ACC can promote the growth of yeast.

[0141] According to some embodiments, the cell culture medium of the present invention is suitable for the growth of prokaryotes. In specific embodiments, the cell culture medium of the present invention is suitable for the growth of microorganisms. In some embodiments, the microorganisms are microbiome microorganisms. In some embodiments, the microorganisms are bacteria. Therefore, in specific embodiments, the cell culture medium of the present invention is suitable for the growth of bacteria. In some embodiments, the bacteria are Escherichia coli or probiotics, such as bacteria of the genus Bifidobacterium and Lactobacillus. Other examples of probiotic species strains are Lactobacillus Paracasei, Bifidobacterium Longum, Lactobacillus Johnsonii, Lactobacillus Fermentum, Pediococcus Acidlacti, such as: Lactobacillus Acidophilus, Lactobacillus Rhamnosus GG, Lactobacillus Helveticus, Bifidobacterium Infantis, Bifidobacterium Lactis, Lactobacillus Bulgaricus, Lactobacillus Silivarius, Lactobacillus Plantarum, Lactobacillus Reuteri, Lactobacillus casei, Lactobacillus salivarius ... Casei, Bifidobacterum Bifidum, Saccharomyces Boulardii, Streptococcus Thermophilus, Bifidobacterum Breve, Bacillus Coagulans and Lactobacillus Brevis. According to some embodiments, the culture medium suitable for bacterial growth is selected from LB and M9. In one embodiment, the cell culture medium supplemented with stable ACC can promote the growth of bacteria.

[0142] According to some embodiments, the cell culture medium of the present invention is suitable for the growth of Archaea. In one embodiment, the cell culture medium supplemented with stabilized ACC is capable of promoting the growth of Archaea.

[0143] According to any aspect and any one embodiment of the present invention, ACC is stabilized by at least one stabilizer. The terms "stabilizing agent" and "stabilizer" are used interchangeably herein, and refer to any substance that helps to maintain calcium carbonate in an amorphous state during ACC production, preparation, storage and / or use. In certain embodiments, stabilizer is a single agent. In other embodiments, the use of several stabilizers is covered. The terms "stable ACC" and "ACC stabilized by at least one stabilizer" can be used interchangeably in some embodiments.

[0144] The stabilizer may comprise a molecule having one or more functional groups selected from, but not limited to, hydroxyl, carboxyl, ester, amine, phosphino, phosphono, phosphate, sulfonyl, sulfate or sulfinyl groups. The hydroxyl-containing compound is combined with a hydroxide, optionally with other functional groups, such as carboxyl, etc., but the hydroxyl group is not esterified.

[0145] According to some embodiments, the stabilizer has low or no toxicity to mammalian cells or organisms, in particular to humans. According to some embodiments, the stabilizer is of food, nutraceutical or pharmaceutical grade.

[0146] In certain embodiments, the ACC stabilizer is independently an organic acid at each occurrence; a phosphorylated, phosphonated, sulfated or sulfonated organic compound; a phosphate or sulfate ester of a hydroxycarboxylic acid; an organic amine compound; an organic compound containing a hydroxyl group; an organic phosphorus compound or a salt thereof; phosphorylated amino acids and their derivatives; bisphosphonates; an organic phosphate compound; an organic phosphonate compound; an organic polyphosphate, an inorganic polyphosphate, an inorganic phosphorous acid, an organic compound having a plurality of functional groups as defined above; an inorganic phosphate and polyphosphate compound; an organic compound having a polyphosphate chain; an organic surfactant; an inorganic ion essential to biology; a carbohydrate and its derivatives, a protein, a phosphorylated protein, a natural and synthetic biopolymer and its derivatives or any combination thereof. According to some embodiments, the stabilizer may also be pharmaceutically active, such as a bisphosphonic acid or ATP.

[0147] Thus, in one embodiment, the stabilizer is selected from the group consisting of: polyphosphates, such as inorganic polyphosphates, organic acids, phosphorylated, phosphonated, sulfated or sulfonated organic compounds, phosphates or sulfates of hydroxycarboxylic acids, phosphorylated amino acids, bisphosphonates, organic polyphosphates, carbohydrates and their derivatives, proteins, peptides, phosphorylated proteins, phosphorylated peptides and any combination thereof. According to another embodiment, the stabilizer is selected from the group consisting of: phosphoserine, adenosine triphosphate, adenosine diphosphate, phytic acid, citric acid, etidronic acid, pyrophosphate, polyphosphate, triphosphate, ethanol, hexametaphosphate, chitin and any combination thereof.

[0148] According to some embodiments, the stabilizer is an organic acid. According to certain embodiments, the organic acid is selected from ascorbic acid, citric acid, lactic acid or acetic acid, oxalic acid, malonic acid, glutaric acid, succinic acid, maleic acid, lactic acid, glutamic acid, aconitic acid, and optionally includes a compound having at least two carboxyl groups, optionally with a molecular weight of not more than 250 g / mol, such as citric acid, tartaric acid, malic acid, etc. According to a specific embodiment, the stabilizer is citric acid.

[0149] In another embodiment, the phosphate of the hydroxycarboxylic acid is phosphoenolpyruvic acid. In another embodiment, the phosphate or sulfate of the hydroxycarboxylic acid comprises an amino acid. Examples of such esters are phosphoserine, phosphothreonine, sulphoserine, sulphatethreonine and phosphocreatine.

[0150] In another embodiment, the stabilizer is a carbohydrate. According to one embodiment, the carbohydrate is selected from monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides, such as sucrose, mannose, glucose, chitosan and chitin. The stabilizer can be a polyol such as glycerol in some embodiments. According to another embodiment, the stabilizer is an amino acid such as serine or threonine. Each possibility represents a separate embodiment of the present invention.

[0151] Non-limiting examples of natural and synthetic biopolymers and derivatives are polynucleotides and glycoproteins.

[0152] Some specific non-limiting examples of such ACC stabilizers found in natural foods or humans that are approved for use in food consumables include phytic acid, citric acid, disodium hydrogen pyrophosphate, adenosine 5′-monophosphate (AMP) sodium salt, adenosine 5′-diphosphate (ADP) sodium salt and adenosine 5′-triphosphate (ATP) disodium salt hydrate, phosphoserine, phosphorylated amino acids, food-grade surfactants, sodium stearoyl lactylate, and combinations thereof.

[0153] According to some embodiments, the stabilizer comprises at least one component selected from the following: a phosphate or sulfate ester of a hydroxycarboxylic acid, such as phosphoenolpyruvate, phosphoserine, phosphothreonine, sulfoserine or sulfothreonine, and a carbohydrate selected from monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides, such as sucrose, mannose, glucose. The hydroxyl-containing organic compound may also include at least one alkali metal hydroxide, such as sodium hydroxide, potassium hydroxide, and the like. The phosphorylated acid may be present as an oligopeptide and a polypeptide. In other embodiments of the present invention, the stabilizer is an organic acid selected from a monocarboxylic acid or a polycarboxylic acid, such as a dicarboxylic acid or a tricarboxylic acid. Each possibility represents a separate embodiment of the present invention. The organic acid may be as defined above.

[0154] In some embodiments of the present invention, ACC stabilizers are selected from phosphorylated amino acids, polyols and combinations thereof. In some embodiments, stable ACC comprises phosphorylated compounds as stabilizers, wherein phosphorylation is performed on the hydroxyl groups of organic compounds. In some embodiments, stable ACC comprises stabilizers selected from the group consisting of: citric acid, phosphoserine, phosphothreonine and combinations thereof. Non-limiting examples of stabilizers comprising phosphate, phosphite, phosphonate groups and their salts or esters include phytic acid, dimethyl phosphate, trimethyl phosphate, sodium pyrophosphate, tetraethyl pyrophosphate, ribulose diphosphate, etidronic acid and other medicinal bisphosphonic acids, 3-phosphoglycerate, 3-phosphoglyceraldehyde, 1-deoxy-D-xylulose-5-phosphate sodium salt, diethylenetriamine penta (methylphosphonic acid), nitrile tri (methylphosphonic acid), 5-phospho-D-ribose 1-diphosphate pentasodium salt, adenosine 5'-diphosphate sodium salt, adenosine 5'-triphosphate disodium salt hydrate, α-D-galactosamine 1-phosphate, 2-phospho-L-ascorbic acid trisodium salt, α-D-galactose 1-phosphate dipotassium salt pentahydrate, α-D-galactosamine 1-phosphate, O-phosphoethanolamine disodium salt hydrate, 2,3-diphospho-D-glyceric acid pentasodium salt, phospho(enol)pyruvate monosodium salt hydrate, D-glyceraldehyde 3-phosphate, sn-glycerol 3-phosphate lithium salt, D-(-)-3-phosphoglyceric acid disodium salt, D-glucose-6-phosphate sodium salt, phosphatidic acid, ibandronate sodium salt, phosphonoacetic acid, DL-2-amino-3-phosphonopropionic acid or a combination thereof. The biologically essential inorganic ions may include, among others, Na, K, Mg, Zn, Fe, P, S, N; P or S in oxide phase; or N as an amine or nitro group.

[0155] Stabilizers may also include phosphonate compounds such as, but not limited to, bisphosphonates, polyphosphates such as, but not limited to, pyrophosphates or polyphosphonates or organic polyphosphates such as, but not limited to, adenosine diphosphate (ADP) or adenosine triphosphate (ATP).

[0156] Optionally, ACC is stabilized by a combination of phosphoserine and citric acid. In another embodiment, ACC is stabilized by triphosphate and citric acid.

[0157] ACC can be stabilized by more than one stabilizer, for example two stabilizers. Stable ACC can include more than two stabilizers, wherein one or more stabilizers are added to the ACC during the formation and precipitation of the ACC; thereby constituting an "internal" stabilizer, and another one or more stabilizers are added to the surface of the ACC particles after the ACC is formed; thereby constituting an "external" stabilizer. Additional examples of stabilized ACC and its preparation can be found in international patent applications WO 2009 / 053967, WO 2014 / 024191 and WO 2016 / 193982.

[0158] In some embodiments, the stabilizer is a protein. In one embodiment, the protein is a naturally produced and purified protein. In another embodiment, the protein is a synthetically produced protein. In some embodiments, the protein is selected from GAP65, GAP22, GAP21 and GAP12 proteins. In another embodiment, the protein is selected from CqCDA1, chitinase 2, β-N-acetylglucosaminidase, GAMP-like, chitin binding protein, CqCBP, CAP10, GAP 18.2, GAP 02526, CqHc1, CqHc2, CqHc3, CqHc4, CqHc5, CqHc6, CqHc7, cryptocyanin 1, cyclophilin, cystatin 1, cycstatin 2, LPS-BP, LEA protein and crystal anthocyanin, optionally from C. quadricarinatus. According to certain embodiments, the protein is a phosphorylated protein.

[0159] In some embodiments, the stabilizer is selected from the group consisting of polyphosphates, phosphorylated amino acids, organic acids, phosphorylated, phosphonated, sulfated or sulfonated organic compounds, phosphates or sulfates of hydroxycarboxylic acids, bisphosphonates, carbohydrates, derivatives thereof, proteins, phosphorylated proteins, natural and synthetic biopolymers and derivatives thereof, and any combination thereof. In other embodiments, the stabilizer is selected from the group consisting of phosphoserine, triphosphates, adenosine triphosphate, adenosine diphosphate, phytic acid, citric acid, etidronic acid, pyrophosphate, ethanol, hexametaphosphate, chitin and any combination thereof.

[0160] In some embodiments, the stabilizer is selected from an organic acid, a phosphorylated organic acid, a phosphate or sulfate ester of a hydroxycarboxylic acid, a phosphorylated amino acid, a bisphosphonate, an organic polyphosphate, a carbohydrate, a derivative thereof, a protein, and any combination thereof.

[0161] According to some embodiments, the at least one stabilizer is selected from the group consisting of polyphosphates, bisphosphonates, phosphorylated amino acids, citric acid, and any combination thereof. In some embodiments, more than one stabilizer is added, such as 2, 3 or 4 stabilizers.

[0162] According to some embodiments, the stabilizer is polyphosphoric acid or a pharmaceutically acceptable salt thereof. According to some embodiments, polyphosphate is a physiologically compatible water-soluble polyphosphate selected from the group consisting of any other necessary cations of sodium, potassium and polyphosphate. In one embodiment, polyphosphate is an organic or inorganic polyphosphate. The term "polyphosphate" as used herein refers to a polymeric ester of PO4. According to some embodiments, polyphosphate is a physiologically compatible water-soluble polyphosphate selected from the group consisting of sodium polyphosphate and potassium polyphosphate. In some embodiments, polyphosphoric acid / polyphosphate is an inorganic polyphosphoric acid or a pharmaceutically acceptable salt thereof. Non-limiting examples of such salts are Na, K, Mg, Mn and Zn. According to some embodiments, inorganic phosphates include 2 to 10 phosphate groups, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate groups. According to some embodiments, polyphosphate is selected from pyrophosphate, triphosphate and hexametaphosphate. According to one embodiment, the stabilizer is pyrophosphoric acid or a pharmaceutically acceptable salt thereof, such as sodium pyrophosphate. According to another embodiment, the stabilizer is triphosphoric acid or a pharmaceutically acceptable salt thereof, such as sodium triphosphate. The terms "triphosphoric acid / triphosphate" and "tripolyphosphoric acid / tripolyphosphate" are used interchangeably herein. According to another embodiment, the stabilizer is hexametaphosphoric acid or a pharmaceutically acceptable salt thereof, such as sodium hexametaphosphate.

[0163] According to some embodiments, the stabilizer is a bisphosphonic acid or a pharmaceutically acceptable salt thereof. Non-limiting examples of salts are Na, K, Mg, Mn and Zn.

[0164] As used herein, the term "bisphosphonic acid / bisphosphonate" refers to an organic compound having two phosphonate (PO(OH)2) groups. The term also relates to compounds having a PO3-organic-PO3 skeleton. The most typical are a series of bisphosphonic acids used as drugs for the treatment of osteoporosis. According to some embodiments, the bisphosphonic acid / bisphosphonate is selected from the group consisting of etidronic acid, zoledronic acid, medronic acid, alendronic acid, and pharmaceutically acceptable salts thereof. According to some embodiments, the stabilizer is etidronic acid or a pharmaceutically acceptable salt thereof. According to another embodiment, the stabilizer is zoledronic acid or a pharmaceutically acceptable salt thereof. According to additional embodiments, the stabilizer is medronic acid or a pharmaceutically acceptable salt thereof. According to certain embodiments, the stabilizer is alendronic acid or a pharmaceutically acceptable salt thereof.

[0165] According to certain embodiments, the stabilizer is a phosphorylated amino acid. According to one embodiment, the phosphorylated amino acid is phosphoserine. According to another embodiment, the phosphorylated amino acid is phosphothreonine.

[0166] According to some embodiments, the ACC composition comprises a combination of stabilizers disclosed above.

[0167] According to some embodiments, the stabilizer is a polyphosphate or diphosphate as defined above, and the molar ratio between the P atoms of the stabilizer and the Ca atoms of the ACC (P:Ca molar ratio) is about 1:90 to 1:1. In one embodiment, the P:Ca molar ratio is about 1:40 to about 1:1. In other embodiments, the P:Ca molar ratio is about 1:35 to about 1:2. In certain embodiments, the P:Ca molar ratio is about 1:30 to about 1:3. In certain embodiments, the P:Ca molar ratio is about 1:28 to about 1:3. In other embodiments, the P:Ca molar ratio is about 1:25 to about 1:4. In other embodiments, the P:Ca molar ratio is about 1:20 to about 1:5. In another embodiment, the P:Ca molar ratio is about 1:20 to about 1:6. In a specific embodiment, the P:Ca molar ratio is about 1:15 to about 1:5. In another specific embodiment, the P:Ca molar ratio is about 1:25 to about 1:5. According to some embodiments, such polyphosphate / polyphosphate is pyrophosphate, triphosphate, hexametaphosphate or a pharmaceutically acceptable salt thereof. According to another embodiment, the bisphosphonic acid / bisphosphonate is alendronic acid, etidronic acid, zoledronic acid or methylenephosphonic acid, and the P:Ca molar ratio is as defined above.

[0168] According to some embodiments, the calcium content (Ca content) of the stabilized ACC comprising polyphosphate or bisphosphonate is from about 1 wt% to about 39 wt%, from about 5 wt% to about 39 wt%, from about 10 wt% to about 39 wt%, from about 15 wt% to about 39 wt%, from about 20 wt% to about 38 wt%, from about 25 wt% to about 38 wt%, or from about 30 wt% to about 38 wt%. The terms "Ca content" and "calcium content" are used interchangeably herein and refer to the calcium content of ACC in the final composition.

[0169] In certain embodiments, the P:Ca molar ratio is about 1:40 to about 1:1, and the Ca content is about 20 wt% to about 39 wt%. In some embodiments, the P:Ca molar ratio is about 1:28 to about 1:3, and the Ca content is about 30 wt% to about 38 wt%. In another embodiment, the molar ratio is 1:25 to about 1:5, and the Ca content is about 30 wt% to about 36 wt%.

[0170] According to some embodiments, the stabilizer is selected from the group consisting of: polyphosphates, phosphorylated amino acids, bisphosphonates, citric acid, tartaric acid, and any combination thereof. According to one embodiment, the polyphosphate is selected from the group consisting of triphosphates, pyrophosphates, and hexametaphosphates, the phosphorylated amino acid is phosphoserine or phosphothreonine, and the bisphosphonate / bisphosphonate is selected from the group consisting of alendronate, etidronic acid, zoledronic acid, and methylenephosphonic acid.

[0171] According to some embodiments, the stabilized ACC comprises less than 20 wt%, less than 15 wt%, less than 10 wt%, or less than 5 wt% of stabilizer. In some embodiments, the stabilized ACC comprises up to 5 wt% of stabilizer.

[0172] According to one embodiment, the average diameter of the primary particles of stable ACC is from about 10nm to about 5μm. According to another embodiment, the average diameter of the primary particles of ACC is from about 30nm to about 400nm. According to yet another embodiment, the average diameter of the primary particles of ACC is from about 30nm to 350nm. According to certain embodiments, the average diameter of the primary particles of ACC is from about 35nm to 300nm, 40nm to about 250nm, about 45nm to about 200nm, about 50nm to about 150nm, or about 60nm to about 100nm. According to still another embodiment, the primary particles of ACC are aggregated, and the average diameter of the aggregates is between 0.5μm and 300μm. According to an additional embodiment, the diameter of the aggregates of the primary particles of ACC is from about 1 to about 100μm, from about 10 to about 50μm, or from about 20 to about 40μm. According to another embodiment, the average diameter of the aggregates of the primary particles of ACC is between 1μm and 10μm.

[0173] According to any one of the above embodiments, the final concentration of stable ACC in the cell culture medium is about 0.1 to about 20mM, about 0.5 to about 15mM, about 1 to about 10mM, about 2 to about 8mM, about 3mM to about 6mM or about 4mM to about 5mM. In a more specific embodiment, stable ACC is present at a concentration of 0.5 to about 4mM, about 1 to about 3mM, about 1.5 to about 2.5 or about 1, 1.5, 2 or 2.5mM. According to some embodiments, the concentration of stable ACC in the cell culture medium is about 0.0001% w / v to about 1% w / v, about 0.0005% w / v to about 0.5% w / v, about 0.001% w / v to about 0.1% w / v, about 0.005% w / v to about 0.05% w / v, about 0.01% w / v to about 0.03% w / v.

[0174] According to some more specific embodiments, the present invention provides a cell culture medium supplemented with ACC stabilized by at least one stabilizer, wherein the stabilizer is selected from polyphosphates, such as inorganic polyphosphates, phosphorylated amino acids, bisphosphonates, organic acids, and any combination thereof; and the cell culture medium is suitable for (i) muscle, nerve or bone cells or tissue culture; (ii) human or non-human mammal embryos; (iii) stem cells; (iv) gamete cells or (v) ovary growth. According to some embodiments, the stabilizer is phosphoserine. According to another embodiment, the stabilizer is a triphosphate. According to yet another embodiment, the stabilizer is a combination of phosphoserine and an organic acid such as citric acid. According to another embodiment, the stabilizer is a combination of triphosphates such as sodium triphosphate and an organic acid such as citric acid. According to one embodiment, such a cell culture medium is suitable for the growth of muscle, nerve or bone cells or tissue culture. According to a more specific embodiment, the cell culture medium can support and promote the growth of cells, such as proliferation, maturation, development or differentiation. According to one embodiment, the cell is a neural cell, and more specifically a damaged neural cell. Therefore, according to one embodiment, such a cell culture medium can promote the regeneration of damaged neural cells. According to another embodiment, the cell is a muscle cell, particularly a malnourished muscle cell, such as a cell of Duchenne muscular dystrophy. Therefore, according to one embodiment, the cell culture medium can promote the formation of myotubes and / or the onset of contractility. According to another embodiment, such a cell culture medium is suitable for the growth of human or non-human mammal embryos. In a specific embodiment, the cell culture medium is suitable for the growth of human embryos. In even more specific embodiments, the cell culture medium can promote the development of human embryos. According to another embodiment, such a cell culture medium is suitable for the growth of human or non-human mammal gamete cells. In a specific embodiment, the cell culture medium is suitable for the maturation of human gamete cells. In even more specific embodiments, the cell culture medium can promote the maturation of human gamete cells. According to another embodiment, the cell culture medium can mature human sperm, such as promoting sperm motility, promoting sperm forward motility, increasing sperm count and any combination thereof. According to other embodiments, such a cell culture medium culture is suitable for stem cells, particularly the growth of human stem cells. According to some embodiments, stem cells are selected from embryonic stem cells, chorionic stem cells, amniotic membrane stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, glial stem cells, adult stem cells and induced pluripotent stem cells. According to one embodiment, such a cell culture medium is suitable for the proliferation and / or differentiation of stem cells. According to a specific embodiment, such a cell culture medium can promote the differentiation of stem cells such as MBA13 into osteoblasts. According to some embodiments, the final concentration of stable ACC in the cell culture medium is about 0.1 to about 8mM, about 0.5 to about 6mM, about 1 to about 5mM or about 2 to about 4mM.According to one embodiment, the cell culture medium supplemented with stable ACC can increase growth parameters, such as proliferation, maturation, development or differentiation of cells by about 10% to about 600%, about 20% to about 500%, about 30% to about 400%, about 40% to about 300%, about 50% to about 200%, about 60% to about 150% or about 70% to about 100%. According to some embodiments, growth parameters are increased by about 100% to about 500%, about 120% to about 400%, about 150% to about 300%.

[0175] According to some embodiments, the invention provides a cell culture medium, such as a cleavage, monoculture or continuous culture medium, supplemented with ACC stabilized by at least one stabilizing agent, wherein the agent is phosphoserine or sodium triphosphate, optionally in combination with citric acid, the final concentration of the stabilized ACC in the cell culture medium is about 0.1 to about 8 mM, about 0.5 to about 6 mM, about 1 to about 5 mM or about 2 to about 4 mM, and the cell culture medium is capable of promoting the growth, such as development, of a human embryo.

[0176] According to some embodiments, the present invention provides a cell culture medium supplemented with ACC stabilized by at least one stabilizer, such as a culture medium for sperm isolation, washing or maturation, e.g. PureCeption TM 、Multipurpose Handling Quinns TM Sperm wash medium and modified HTF medium with gentamicin, wherein the agent is phosphoserine or sodium triphosphate, optionally in combination with citric acid, the final concentration of stable ACC in the cell culture medium is about 0.1 to about 8 mM, about 0.5 to about 6 mM, about 1 to about 5 mM or about 2 to about 4 mM, and the cell culture medium is capable of promoting the maturation of sperm cells, such as promoting sperm motility, promoting sperm progressive motility, increasing sperm count, and any combination thereof.

[0177] According to other embodiments, the present invention provides a cell culture medium supplemented with ACC stabilized by at least one stabilizing agent, wherein the agent is phosphoserine, etidronic acid or sodium triphosphate, optionally in combination with citric acid, and the cell culture medium is capable of promoting the regeneration of neural cells.

[0178] According to other embodiments, the present invention provides a cell culture medium, such as DMEM / F12 or DMEM / F12 optionally supplemented with horse serum (HS), L-glutamine, gentamicin and insulin, wherein the culture medium is further supplemented with ACC stabilized by phosphoserine, etidronic acid or sodium triphosphate, optionally in combination with citric acid, wherein the final concentration of the stabilized ACC in the cell culture medium is about 0.1 to about 8 mM, about 0.5 to about 6 mM, about 1 to about 5 mM or about 2 to about 4 mM, and the cell culture medium is capable of promoting the formation of myotubes and / or the onset of contractility in skeletal muscle cells, for example in the case of Duchenne muscular dystrophy.

[0179] According to other embodiments, the present invention provides a cell culture medium supplemented with ACC stabilized by phosphoserine, etidronic acid or sodium triphosphate, optionally in combination with citric acid, and the cell culture medium is capable of promoting the differentiation of stem cells, in particular the differentiation of MBA13 into osteoblasts, wherein the final concentration of the stabilized ACC in the cell culture medium is about 0.1 to about 8 mM, about 0.5 to about 6 mM, about 1 to about 5 mM or about 2 to about 4 mM.

[0180] According to other embodiments, the present invention provides a cell culture medium, such as DMEM / F12 or DMEM / F12 with 10% fetal bovine serum (FBS), 2mM glutamine, 25μg / mL gentamicin and 0.3-0.5% NVR-Gel, wherein the culture medium is further supplemented with ACC stabilized by phosphoserine, etidronic acid or sodium triphosphate, optionally in combination with citric acid, wherein the final concentration of the stabilized ACC in the cell culture medium is about 0.1 to about 8mM, about 0.5 to about 6mM, about 1 to about 5mM or about 2 to about 4mM, and the cell culture medium is capable of promoting the preservation of ovaries.

[0181] According to another aspect, the present invention provides amorphous calcium carbonate (ACC) stabilized by at least one stabilizer for use as a supplement for a cell culture medium. The terms "supplement" and "cell culture medium supplement" are used interchangeably herein and refer to one or more components for adding to a cell culture medium. In a specific embodiment, the term refers to a stable ACC added to or intended to be added to a cell culture medium. According to one embodiment, the stable ACC is added to the culture medium during culture medium preparation. According to another embodiment, ACC is added to the culture medium before use.

[0182] According to one embodiment, the stabilized ACC is for use as a supplement to a cell culture medium suitable for growing a culture of an organism.

[0183] According to some embodiments, when added to a cell culture medium, the stable ACC of the present invention promotes the growth of cells. Therefore, in one embodiment, the stable ACC used as a cell culture medium supplement can promote the growth of cells. In one embodiment, the stable ACC used as a cell culture medium supplement promotes the proliferation, maturation, reproduction, regeneration, development and / or differentiation of cells, tissues and organs. According to one embodiment, the ACC of the present invention used as a cell culture medium supplement can promote the differentiation of stem cells. According to another embodiment, such ACC used as a cell culture medium supplement can promote the proliferation of cells. According to yet another embodiment, the ACC used as a cell culture medium supplement can promote maturation, such as the maturation of gametes, or promote the development of cells, such as the development of embryos. According to certain embodiments, the ACC used as a cell culture medium supplement can promote the regeneration of cells.

[0184] According to one embodiment, the cell culture medium supplemented with stable ACC can increase growth parameters, such as proliferation, maturation, development or differentiation of cells by about 10% to about 600%, about 20% to about 500%, about 30% to about 400%, about 40% to about 300%, about 50% to about 200%, about 60% to about 150% or about 70% to about 100%. According to some embodiments, growth parameters are increased by about 100% to about 500%, about 120% to about 400%, about 150% to about 300%.

[0185] According to some embodiments, the cell culture medium supplement can be added to any known cell culture medium. According to certain embodiments, the culture medium is as defined above. According to one embodiment, the cell culture medium is selected from a natural culture medium and an artificial culture medium. According to one embodiment, the culture medium is suitable for the growth of any biological culture, such as cell culture, tissue culture, organ culture, unicellular eukaryotic organisms or microorganisms such as bacteria. According to one embodiment, the cell culture medium is suitable for the growth of a culture selected from a culture of animal, plant or insect cells, tissues and / or organs. According to another embodiment, the cell culture medium is suitable for the growth of bacteria or yeast.

[0186] According to some embodiments, the culture of animal cells is selected from the cell culture, tissue culture, organ culture, stem cell, gamete, embryo and organ of human or non-human mammal. According to other embodiments, mammal is non-human mammal, such as livestock animal, such as cattle, pig, sheep, goat, horse, mule, donkey, buffalo or camel; domestic pet, such as cat or dog; rodent, such as mouse, rat, guinea pig or hamster; lagomorph, such as rabbit; or primate, such as monkey (such as macaque) or ape (such as chimpanzee). According to some embodiments, stable ACC is used as a supplement for cell culture medium suitable for primary or passage culture growth. According to some embodiments, stable ACC can promote the growth of the cell line. According to some embodiments, stable ACC is used as a supplement for cell culture medium suitable for mammalian tissue culture growth. According to some embodiments, stable ACC is used as a supplement for cell culture medium suitable for stem cell growth. According to another embodiment, stem cells are human or non-human mammal stem cells.

[0187] According to some embodiments, stable ACC is used as a supplement for a cell culture medium suitable for embryonic growth. According to one embodiment, the cell culture medium of the present invention is suitable for embryonic maturation and development. According to another embodiment, the stable ACC used as a supplement for a cell culture medium can promote embryonic development. According to one embodiment, the embryo is a human embryo. According to another embodiment, the embryo is a non-human mammalian embryo.

[0188] According to some embodiments, the stabilized ACC is used as a supplement for a cell culture medium suitable for gamete growth. According to one embodiment, the cell culture medium of the present invention is suitable for maturation or preservation of gametes. According to another embodiment, the stabilized ACC for use as a supplement for a cell culture medium can promote maturation or preservation of gametes, such as maturation or preservation of sperm or oocytes.

[0189] According to another embodiment, the stabilized ACC for use as a supplement for cell culture medium is capable of facilitating cryopreservation of embryos or gametes, as described above.

[0190] According to some embodiments, promoting the maturation of sperm is selected from promoting sperm motility, promoting sperm forward motility, increasing sperm count and any combination thereof. According to one embodiment, sperm is human sperm. According to another embodiment, sperm is non-human mammalian sperm. According to some embodiments, stable ACC is used as a supplement for cell culture medium suitable for organ culture or organ growth. According to some embodiments, organ tissue or organ is selected from ovary, cornea, heart, kidney, pancreas, liver, spleen, lung, testis, bladder and blood vesicle. In a specific embodiment, organ tissue or organ is ovary.

[0191] According to some embodiments, the stabilized ACC is for use as a supplement in a cell culture medium suitable for growing plant or insect cells.

[0192] According to some embodiments, the stabilized ACC is used as a supplement for a cell culture medium suitable for growing animal, plant or insect cells, wherein the cell culture medium is a natural culture medium selected from biological fluids, tissue extracts and clots; or an artificial culture medium selected from balanced salt solutions, basal media and complex media.

[0193] According to some embodiments, the stabilized ACC is for use as a supplement to a cell culture medium selected from the group consisting of DMEM, RPMI 1640, MEM, IMDM, L-15 medium (Leibovitz), MCDB medium, medium 199, opti-MEM and DMEM / F-12, Schneider Drosophila medium, Grace insect medium, IPL-41 insect medium Sf-900, serum-free insect medium, Shields and Sang M3 insect medium, TC-100 insect medium, TNM-FH insect medium, Ham's F-12, Ham's F-10, GMEM, Ames medium, Eagle's basic medium (BME), Claycomb, Click medium, Glasgow's minimum essential medium (GMEM), MegaCell medium, McCoy's 5A modified medium, NCTC medium, Williams medium E, Waymouth medium, TC-10 and IPL-10 medium. According to one embodiment, the stabilized ACC is used as a supplement for a cell culture medium selected from a monoculture medium such as Quinn's Advantage TM (SAGE) Medium 1-Step TM , continuous media, such as Quinn's Advantage TM, continuous medium (ORIGIO), MEDICULT, universal IVM, DMEM, RPMI 1640, MEM, IMDM, opti-MEM, GMEM, Ham's F-12, DMEM / F-12, Schneider Drosophila medium, Grace insect medium, Sf-900, TC-10, IPL-10, B5, N6 and Nitsch medium. According to some embodiments, the stabilized ACC is used as a supplement to a cell culture medium selected from a culture medium for sperm, a culture medium for sperm or a maturation medium, for example. PureCeption TM 、Multipurpose Handling Quinns TM Sperm washing medium and modified HTF medium with gentamicin; medium for fertilization; medium for embryo development and medium for embryo and / or gamete maturation, processing and / or cryopreservation.

[0194] According to these embodiments, ACC for use as a supplement for cell culture medium is capable of promoting the growth of cell culture, tissue culture or organ culture when added to cell culture medium.

[0195] According to some embodiments, stable ACC is used as a supplement for a cell culture medium suitable for the growth of a unicellular eukaryotic organism. According to one embodiment, the unicellular eukaryotic organism is a yeast such as a yeast, more specifically a cerevisiae yeast. According to one embodiment, stable ACC is used as a supplement for YPD, YPG or YPAD culture medium.

[0196] According to some embodiments, stable ACC is used as a supplement for cell culture media suitable for prokaryotes such as bacterial growth. In some embodiments, the bacteria are Escherichia coli or probiotics, such as bacteria of the genus Bifidobacterium and Lactobacillus. According to one embodiment, stable ACC is used as a supplement for LB and M9 culture media.

[0197] According to these embodiments, the ACC for use as a supplement for a cell culture medium is capable of promoting the growth of the yeast or the bacteria when added to the cell culture medium.

[0198] According to some embodiments, the stabilized ACC is for use as a supplement in a cell culture medium suitable for the growth of archaea.

[0199] According to some embodiments, the stabilizer is selected from the group consisting of polyphosphates such as triphosphates, phosphorylated amino acids such as phosphoserine, diphosphates, citric acid, and any combination thereof such as a combination of triphosphate and citric acid or a combination of phosphoserine and citric acid.

[0200] According to some embodiments, the stable ACC for use as a supplement to a cell culture medium remains stable in a dry form in an amorphous phase for a period of at least 7 days, at least 1 month, at least 3 months, at least 6 months, or at least 1 year. In other embodiments, the stable ACC remains stable in an amorphous phase when dispersed in a cell culture medium for a period of at least 1 hour, at least 4, 6, 8, 12 or 24 hours, at least 7 days, or at least 1 month.

[0201] According to any one of the above-mentioned embodiments, the stable ACC used as a supplement for cell culture medium can be formulated into solid, liquid or semi-liquid form. According to some embodiments, the stable ACC used for the present invention is formulated into a solid form, such as a powder, tablet, capsule or granule. In a specific embodiment, the stable ACC used as a supplement is formulated into a powder. According to some embodiments, the stable ACC used as a supplement is formulated into a liquid or semi-liquid form. According to one embodiment, the liquid form is a suspension or an emulsion, and the semi-liquid form is a gel or a viscous suspension, such as a colloidal suspension. In a specific embodiment, the stable ACC is formulated into a suspension. Therefore, in one embodiment, during the preparation of the cell culture medium or before using the culture medium, the stable ACC used as a supplement for the cell culture medium is added to the cell culture medium with a powder or a suspension. According to an even more specific embodiment, the stable ACC is added as a freshly prepared suspension.

[0202] According to one embodiment, the average diameter of the primary particles of the stable ACC is about 10 nm to about 5 μm. According to another embodiment, the average diameter of the primary particles of ACC is about 30 nm to about 400 nm. According to yet another embodiment, the average diameter of the primary particles of ACC is about 30 nm to 350 nm.

[0203] According to certain embodiments, the average diameter of the primary particles of ACC is about 35nm to 300nm, 40nm to about 250nm, about 45nm to about 200nm, about 50nm to about 150nm, or about 60nm to about 100nm. According to still another embodiment, the primary particles of ACC are aggregated, and the average diameter of the aggregates is between 0.5μm and 300μm. According to an additional embodiment, the diameter of the aggregates of the primary particles of ACC is about 1 to about 100μm, about 10 to about 50μm, or about 20 to about 40μm. According to another embodiment, the average diameter of the aggregates of the primary particles of ACC is between 1μm and 10μm.

[0204] According to any of the above embodiments, ACC for use as a supplement to a cell culture medium is added to a cell culture medium at a final concentration of about 0.1 to about 20 mM, about 0.5 to about 15 mM, about 1 to about 10 mM, about 2 to about 8 mM, about 3 mM to about 6 mM, or about 4 mM to about 5 mM. In more specific embodiments, stable ACC is present at a concentration of 0.5 to about 4 mM, about 1 to about 3 mM, about 1.5 to about 2.5, or about 1, 1.5, 2, or 2.5 mM.

[0205] According to some more specific embodiments, the present invention provides ACC stabilized by at least one stabilizer, the stabilizer selected from polyphosphates, such as organic polyphosphates, phosphorylated amino acids, bisphosphonates, organic acids and any combination thereof, for use as a supplement to a cell culture medium suitable for the growth of (i) muscle, nerve or bone cells or tissue cultures; (ii) human or non-human mammalian embryos; (iii) stem cells; (iv) gametes or (v) ovaries. According to some embodiments, the stabilizer is phosphoserine. According to another embodiment, the stabilizer is a triphosphate. According to yet another embodiment, the stabilizer is a combination of phosphoserine and an organic acid such as citric acid. According to another embodiment, the stabilizer is a combination of triphosphates such as sodium triphosphate and an organic acid such as citric acid. According to one embodiment, such a cell culture medium is suitable for the growth of muscle, nerve or bone cells or tissue cultures. According to one embodiment, the stable ACC supplemented with the cell culture medium can promote the growth of cells, such as proliferation, development, maturation or differentiation. According to one embodiment, the cell is a neural cell, and more specifically a damaged neural cell. According to one embodiment, the stable ACC used as a supplement to a cell culture medium can promote the regeneration of damaged nerve cells. According to another embodiment, the cell is a muscle cell, in particular a dystrophic muscle cell, such as a cell of Duchenne muscular dystrophy. According to one embodiment, the stable ACC used as a supplement to a cell culture medium can promote the onset of myotube formation and / or myocyte contractility. According to one embodiment, the stable ACC used as a supplement to a cell culture medium can promote the development of human embryos. According to one embodiment, the stable ACC used as a supplement to a cell culture medium can promote the maturation or preservation of gametes, and in particular sperm. According to another embodiment, the stable ACC used as a supplement to a cell culture medium can promote the cryopreservation of embryos or gametes, as described above. According to one embodiment, the stable ACC used as a supplement to a cell culture medium can promote sperm motility, promote sperm forward motility, increase sperm counts, and any combination thereof. According to another embodiment, the stable ACC used as a supplement can promote the growth of stem cells, and in particular human stem cells, such as proliferation, amplification, and / or differentiation. According to some embodiments, stem cells are selected from embryonic stem cells, chorionic stem cells, amniotic membrane stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, glial stem cells, adult stem cells and induced pluripotent stem cells. According to one embodiment, the cell culture medium supplemented with stable ACC can increase growth parameters, such as cell proliferation, maturation, development or differentiation by about 10% to about 600%, about 20% to about 500%, about 30% to about 400%, about 40% to about 300%, about 50% to about 200%, about 60% to about 150% or about 70% to about 100%.According to some embodiments, the growth parameter is increased by about 100% to about 500%, about 120% to about 400%, about 150% to about 300%.

[0206] According to some embodiments, the present invention provides stable ACC for use as a supplement for cleavage, monoculture or sequential culture media, wherein the stabilizer is phosphoserine or sodium triphosphate, optionally in combination with citric acid, and the stable ACC is capable of promoting the development of human embryos.

[0207] According to some embodiments, the present invention provides stable ACC for use as a supplement for cell culture medium, wherein the agent is phosphoserine, etidronic acid or sodium triphosphate, optionally in combination with citric acid, and the stable ACC is capable of promoting regeneration of neural cells.

[0208] According to some embodiments, the present invention provides stable ACC for use as a supplement to DMEM / F12 or DMEM / F12 optionally supplemented with horse serum (HS), L-glutamine, gentamicin and insulin, wherein the stabilizer is selected from phosphoserine, etidronic acid or sodium triphosphate, optionally in combination with citric acid, and the stable ACC is capable of promoting the formation of myotubes and / or the onset of contractility in skeletal muscle cells, for example in the case of Duchenne muscular dystrophy.

[0209] According to some embodiments, the present invention provides ACC stabilized by phosphoserine, etidronic acid or sodium triphosphate, optionally in combination with citric acid, for use as a supplement for cell culture medium and thereby capable of promoting differentiation of stem cells, in particular differentiation of MBA13 into osteoblasts.

[0210] According to some embodiments, the present invention provides ACC stabilized by phosphoserine, etidronic acid or sodium triphosphate, optionally in combination with citric acid, for use as a supplement for sperm separation, washing or maturation medium, such as PureCeption TM 、Multipurpose Handling Quinns TM Sperm washing medium, modified HTF medium with gentamicin, and thereby capable of promoting the maturation of sperm cells, such as promoting sperm motility, promoting sperm progressive motility, increasing sperm count, and any combination thereof.

[0211] The present invention provides ACC stabilized by phosphoserine, etidronic acid or sodium triphosphate, optionally in combination with citric acid, for use as a supplement for culture media such as DMEM / F12 or DMEM / F12 with 10% fetal bovine serum (FBS), 2mM glutamine, 25μg / mL gentamicin and 0.3-0.5% NVR-Gel, thereby promoting the preservation of ovaries.

[0212] According to some embodiments, the final concentration of the stabilized ACC in the cell culture medium is about 0.1 to about 8 mM, about 0.5 to about 6 mM, about 1 to about 5 mM, or about 2 to about 4 mM.

[0213] According to yet another aspect, the present invention provides a cell culture medium supplement comprising amorphous calcium carbonate (ACC) stabilized by at least one stabilizer.

[0214] According to one embodiment, the cell culture medium supplement comprising stable ACC is added to the culture medium during the preparation of the culture medium. According to another embodiment, the supplement is added to the culture medium before use.

[0215] According to some embodiments, the cell culture medium supplement is a solid supplement. According to other embodiments, the supplement is a liquid or semi-liquid supplement.

[0216] According to any one of the above embodiments, ACC is stabilized by at least one stabilizer as defined above. According to some embodiments, the stabilizer is selected from the group consisting of: polyphosphates such as triphosphates, phosphorylated amino acids such as phosphoserine, diphosphates, citric acid and any combination thereof such as a combination of triphosphates and citric acid or a combination of phosphoserine and citric acid.

[0217] The cell culture medium supplement of the present invention comprising stable ACC can be added to any cell culture medium described above. In one embodiment, the cell culture medium supplement comprising stable ACC can be added to a cell culture medium suitable for growing biological cultures, such as a culture of cells, tissue culture, organ culture or organ. The cell can be a eukaryotic cell or a prokaryotic cell. Specifically, the cell culture medium refers to a culture medium suitable for eukaryotic cell culture, tissue culture or organ growth. According to some embodiments, the cell culture is a suspension or adherent cell culture. In some specific embodiments, the culture medium can be a complete culture medium, a basal culture medium, a basal culture medium supplemented with a cell culture medium supplement, a culture medium with various amounts of serum or a chemically defined culture medium.

[0218] According to some embodiments, the cell culture medium supplement comprising stable ACC can promote the growth of cells or tissues. Therefore, in one embodiment, the cell culture medium supplement comprising stable ACC can promote the proliferation, maturation, reproduction, regeneration, development and / or differentiation of cells, tissues and organs. According to one embodiment, the supplement can promote the differentiation of stem cells. According to another embodiment, the supplement can promote the proliferation of the culture of cells. According to yet another embodiment, the supplement can promote the maturation and / or development of cells or tissues. According to certain embodiments, the supplement can promote the regeneration of cells. According to some embodiments, the cell is a eukaryotic cell. According to one embodiment, the eukaryotic cell is an animal, plant and insect cell. According to another embodiment, the culture of animal cells is selected from the cell culture, tissue culture, organ culture, stem cell, gamete, embryo and organ of a human or non-human mammal. According to some embodiments, the mammal is a human, and the culture of human cells is selected from human cells, tissue culture and organ culture. According to another embodiment, the mammal is a non-human mammal. According to some embodiments, the cell culture, the human or non-human mammal cell culture is selected from the cell culture of nerve, muscle, epithelium, bone, fat, stem cell, gamete and blood cell. According to one embodiment, the cell culture medium supplemented with stable ACC can increase growth parameters, such as proliferation, maturation, development or differentiation of cells by about 10% to about 600%, about 20% to about 500%, about 30% to about 400%, about 40% to about 300%, about 50% to about 200%, about 60% to about 150% or about 70% to about 100%. According to some embodiments, growth parameters are increased by about 100% to about 500%, about 120% to about 400%, about 150% to about 300%.

[0219] According to some embodiments, a cell culture medium supplement comprising stabilized ACC may be added to a cell culture medium suitable for yeast growth, and the cell culture medium is selected from the group consisting of YPD, YPG, and YPAD.

[0220] According to some embodiments, a cell culture medium supplement comprising stabilized ACC may be added to a cell culture medium suitable for the growth of prokaryotes, and the cell culture medium is selected from LB and M9.

[0221] According to some embodiments, the cell culture medium supplement comprising stabilized ACC is capable of promoting the growth of yeast and bacteria.

[0222] According to some more specific embodiments, the present invention provides a cell culture medium supplement comprising ACC stabilized by at least one stabilizer, wherein the agent is selected from phosphorylated amino acids, polyphosphates, bisphosphonates, organic acids, and any combination thereof. According to some embodiments, the supplement is added to a cell culture medium suitable for the growth of (i) muscle, nerve or bone cells or tissue cultures; (ii) human or non-human mammalian embryos; (iii) stem cells; (iv) gametes or (v) ovaries. According to some embodiments, the stabilizer is phosphoserine. According to another embodiment, the stabilizer is a triphosphate. According to yet another embodiment, the stabilizer is a combination of phosphoserine and an organic acid such as citric acid. According to another embodiment, the stabilizer is a combination of triphosphates such as sodium triphosphate and an organic acid such as citric acid. According to a more specific embodiment, the cell culture medium supplement can promote cell growth, such as proliferation, differentiation, development or maturation. According to one embodiment, such a cell culture medium supplement can promote the regeneration of damaged nerve cells. According to another embodiment, such a cell culture medium supplement can promote myotube formation and / or promote the onset of myotube contractility. According to another embodiment, such a cell culture medium supplement can promote the development of embryos such as human embryos. According to another embodiment, such a cell culture medium supplement is capable of promoting the maturation or preservation of gametes and in particular sperm. According to a specific embodiment, as described above, such a cell culture medium supplement is capable of promoting cryopreservation of embryos or gametes. According to one embodiment, such a cell culture medium is suitable for the proliferation and differentiation of stem cells. According to some embodiments, the stem cells are selected from embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, glial stem cells, adult stem cells and induced pluripotent stem cells. According to another embodiment, such a cell culture medium supplement is capable of promoting the differentiation of stem cells. According to a specific embodiment, such a cell culture medium supplement is capable of promoting the differentiation of stem cells such as MBA13 to osteoblasts. According to one embodiment, a cell culture medium supplemented with stable ACC is capable of increasing growth parameters, such as cell proliferation, maturation, development or differentiation by about 10% to about 600%, about 20% to about 500%, about 30% to about 400%, about 40% to about 300%, about 50% to about 200%, about 60% to about 150% or about 70% to about 100%. According to some embodiments, the growth parameter is increased by about 100% to about 500%, about 120% to about 400%, about 150% to about 300%.

[0223] According to some embodiments, the present invention provides a cell culture medium supplement comprising ACC stabilized by at least one stabilizer selected from phosphoserine, etidronic acid or sodium triphosphate, optionally in combination with citric acid. According to one embodiment, the supplement is added to a cleavage, monoculture or continuous culture medium to promote the development of human embryos. According to one embodiment, the supplement is added to a culture medium for sperm separation, washing or maturation, such as PureCeption TM 、Multipurpose Handling Quinns TM Sperm washing medium or modified HTF medium with gentamicin, thereby promoting the maturation or preservation of gametes and, in particular, sperm. According to another embodiment, such a supplement is added to a cell culture medium suitable for the growth of neural cells, thereby promoting the regeneration of neural cells. According to another embodiment, the supplement is added to a DMEM / F12 medium or a DMEM / F12 medium optionally supplemented with horse serum (HS), L-glutamine, gentamicin and insulin, thereby promoting the formation of myotubes in skeletal muscle cells, for example, in the case of Duchenne muscular dystrophy. According to yet another embodiment, the supplement is added to a cell culture medium suitable for the growth of stem cells, thereby promoting the differentiation of stem cells, in particular the differentiation of MBA13 into osteoblasts. According to other embodiments, the supplement is added to a culture medium, such as DMEM / F12 or DMEM / F12 with 10% fetal bovine serum (FBS), 2mM glutamine, 25μg / mL gentamicin and 0.3-0.5% NVR-Gel, thereby promoting the preservation of ovaries. According to some embodiments, the final concentration of the stabilized ACC in the cell culture medium is about 0.1 to about 8 mM, about 0.5 to about 6 mM, about 1 to about 5 mM, or about 2 to about 4 mM.

[0224] According to any of the above embodiments, the term "comprises" has the meaning of "consisting of", thus according to such embodiments, the cell culture medium supplement consists of ACC stabilized by at least one stabilizer.

[0225] According to a further aspect, the present invention provides amorphous calcium carbonate (ACC) stabilized by at least one stabilizer and formulated as a supplement for a cell culture medium.

[0226] According to certain aspects, the present invention provides a method for promoting the growth of a biological culture, the method comprising exposing the culture to ACC stabilized by at least one stabilizer. According to one embodiment, the biological culture is selected from the group consisting of eukaryotic cells, tissues or organs and prokaryotic cells.

[0227] According to other embodiments, the method comprises promoting growth of biological cultures, such as cells, tissues and organs, such as promoting proliferation, maturation, reproduction, regeneration, differentiation and / or development thereof.

[0228] According to one embodiment, the culture of cells is selected from animal, plant or insect cell cultures. According to another embodiment, the tissue culture is selected from animal, plant or insect tissue cultures. According to another embodiment, the organ culture is selected from animal, plant or insect organ cultures. According to some embodiments, the animal is a human or non-human mammal. According to certain embodiments, the non-human mammal is selected from livestock animals, such as cattle, pigs, sheep, goats, horses, mules, donkeys, buffaloes or camels; domestic pets, such as cats or dogs; rodents, such as mice, rats, guinea pigs or hamsters; lagomorphs, such as rabbits; and primates, such as monkeys (e.g., macaques) or apes (e.g., chimpanzees).

[0229] According to some embodiments, the mammalian cell culture, human or non-human mammalian cell culture is selected from the cell culture of nerve, muscle, epithelium, bone, fat, stem cell, gamete and blood cell. According to one embodiment, the tissue culture is selected from the culture of epithelial tissue, connective tissue, muscle tissue and neural tissue. According to some more specific embodiments, the tissue culture is selected from the culture of kidney, liver, gland, brain, bone, eye and muscle tissue. According to some embodiments, the organ tissue or organ is selected from the ovary, cornea, heart, kidney, pancreas, liver, spleen, lung, testis, bladder and blood vesicle. In a specific embodiment, the organ tissue or organ is ovary.

[0230] According to one embodiment, the present invention provides a method for promoting the growth of muscle cells. According to another embodiment, promoting the growth of muscle cells comprises promoting the formation of myotubes. According to some embodiments, the method includes also reducing the time for the onset of spontaneous contractile activity of the myotubes. The time for the onset of spontaneous contractile activity is defined as the time required for myoblasts to fuse and begin spontaneous contraction. In one embodiment, the muscle cells formed by the myoblasts are selected from skeletal muscle cells or cardiomyocytes. In more specific embodiments, the muscle cells are skeletal muscle cells. According to some embodiments, the method includes promoting the formation of myotubes in skeletal muscle cells and / or the onset of contractility, for example in the case of Duchenne muscular dystrophy.

[0231] According to other embodiments, the method includes promoting the growth of nerve cells. In one embodiment, promoting the growth of nerve cells comprises promoting and accelerating nerve cell regeneration. Therefore, in one embodiment, the method includes promoting the regrowth of axons and dendrites neuron fibers of the peripheral and central nervous systems, and / or sprouting from damaged neuron fibers.

[0232] According to some embodiments, the method comprises promoting the maturation or preservation of gametes, such as promoting the in vitro maturation or preservation of sperm or oocytes. According to some embodiments, the gametes are selected from human gametes or non-human mammal gametes. According to one embodiment, the gametes are sperm. Therefore, in one embodiment, the present invention provides a method for promoting sperm maturation or improving sperm quality, the method comprising exposing the culture to ACC stabilized by at least one stabilizer.

[0233] According to one embodiment, improving sperm quality is selected from the group consisting of promoting sperm motility, promoting sperm progressive motility, increasing sperm count, and any combination thereof.

[0234] According to some embodiments, increasing sperm count comprises increasing sperm count in a motility or progressive motility procedure. According to one embodiment, the motility or progressive motility procedure is an upstream procedure.

[0235] According to any of the above embodiments, the sperm is human or non-human mammalian sperm. According to some embodiments, the non-human mammal is selected from the group consisting of livestock animals, domestic pets, rodents, wild animals and primates.

[0236] In one embodiment, the livestock animal is selected from cattle, pigs, sheep, goats, horses, mules, donkeys, buffalo and camels. In some other embodiments, the domestic pet is a cat or dog, the rodent is a rat, mouse, guinea pig or hamster, the lagomorph is a rabbit, and the primate is a monkey such as a macaque or an ape such as a chimpanzee.

[0237] According to another embodiment, the sperm is a non-mammalian sperm. According to some embodiments, the non-mammal is selected from the group consisting of fish, insects and birds.

[0238] According to one embodiment, the sperm is human sperm. According to other embodiments, the method comprises promoting in vitro embryo development.

[0239] According to some embodiments, the method includes promoting differentiation and / or proliferation of stem cells. According to some embodiments, the stem cells are selected from embryonic stem cells, chorionic stem cells, amniotic membrane stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, glial stem cells, adult stem cells and induced pluripotent stem cells. According to one embodiment, the method includes promoting differentiation of stem cells such as MBA13 into osteoblasts.

[0240] According to one embodiment, the cell culture medium supplemented with stable ACC can increase growth parameters, such as proliferation, maturation, development or differentiation of cells by about 10% to about 600%, about 20% to about 500%, about 30% to about 400%, about 40% to about 300%, about 50% to about 200%, about 60% to about 150% or about 70% to about 100%. According to some embodiments, growth parameters are increased by about 100% to about 500%, about 120% to about 400%, about 150% to about 300%.

[0241] According to some embodiments, the culture of cells is a culture of bacteria or yeast, and thus according to such embodiments, the method comprises promoting the growth of yeast or bacteria. In some embodiments, the bacteria is E. coli or a probiotic, such as bacteria of the genera Bifidobacterium and Lactobacillus.

[0242] According to any one of the above embodiments, cells are exposed to ACC stabilized by at least one stabilizer, including adding the ACC to a cell culture medium. The terms "exposed to" and "contacted with..." are used interchangeably herein, referring to placing or transferring cells to a culture medium containing a component of interest (e.g., stable ACC), or adding a component (e.g., stable ACC) to a culture medium in which cells are grown or cultured. The cell culture medium can be any culture medium known in the art. According to certain embodiments, the culture medium is as defined above. According to one embodiment, the cell culture medium is selected from a natural culture medium and an artificial culture medium. According to some embodiments, the natural culture medium comprises a biological fluid selected from plasma, serum, lymph, human placental cord serum, and amniotic fluid. According to another embodiment, the natural culture medium comprises a tissue extract, such as an extract of liver, spleen, tumor, leukocyte, and bone marrow, an extract of bovine embryo and chicken embryo. According to another embodiment, the natural culture medium comprises a coagulant or a plasma clot. According to some embodiments, the culture medium is an artificial culture medium supplemented with ACC stabilized by at least one stabilizer. According to one embodiment, the artificial culture medium is a balanced salt solution. Examples of balanced salt solutions are PBS, DPBS, HBSS, EBSS Tyrode's T6, WM1, Pool's P1, Quinn's HTF and Gardner's G1. According to another embodiment, the artificial culture medium is a basal medium. According to some embodiments, the culture medium may be further supplemented as known in the art. According to one embodiment, the culture medium is supplemented with serum, such as fetal bovine serum. According to another embodiment, the artificial culture medium is a complex culture medium.

[0243] According to one embodiment, the artificial culture medium is a serum-free culture medium. According to a further embodiment, the artificial culture medium is a culture medium with reduced serum content. According to another embodiment, the artificial culture medium is a protein-free culture medium.

[0244] According to one embodiment, the method comprises adding stabilized ACC to a cell culture medium selected from the group consisting of DMEM, EMEM, RPMI 1640 medium, Basal Medium Eagle (BME), Ham's F-10, Ham's F-12, DMEM / F-12, IMDM, opti-MEM, GMEM, IPL-41 insect medium, Schneider Drosophila medium, Grace insect medium, serum-free insect medium, Sf-900, TC-10, Shields and Sang M3 insect medium, TC-100 insect medium, TNM-FH insect medium, IPL-10, monoculture medium, such as Quinn's Advantage TM (SAGE) Medium 1-Step TM or continuous media, such as Quinns Quinn's Advantage TM Cleavage medium, Murashige and Skoog (MS), B5, N6, Nitsch medium, NCTC medium, MegaCell medium, Claycomb, Click medium, L-15 medium, medium 199, MCDB medium, Ames medium, BGJb medium, Click medium, CMRL-1066 medium, McCoy's 5A modified medium, NCTC medium, Swim's S-77 medium, Waymouth medium, Willliam's medium E, in vitro maturation medium, Menezo's B2 and B3 medium, Behr's blastocyst medium, Gardner's G2, universal IVM (ORIGIO) and continuous medium for embryo growth. According to one embodiment, the method comprises adding stable ACC to a cell culture medium selected from a medium for sperm separation, a medium for sperm washing and a medium for maturation, such as PureCeption TM 、Multipurpose Handling Quinns TM Sperm washing medium, modified HTF medium with gentamicin. According to one embodiment, the method comprises adding stable ACC to a cell culture medium selected from a medium for fertilization, a medium for embryo development, or a medium for embryo or / and gamete maturation, processing and / or cryopreservation.

[0245] In some embodiments, cells are grown as stationary or immobilized cultures on supports such as Cytodex 1 or 3, Cytopore 2, polystyrene, gelatin, dextran, polyacrylamide, or other alternative supports.

[0246] According to some embodiments, the cell culture medium is suitable for the growth of unicellular eukaryotic organisms. According to one embodiment, the unicellular eukaryotic organism is a yeast such as a yeast, more specifically a saccharomyces cerevisiae. According to one embodiment, the method includes adding stable ACC to a cell culture medium suitable for the growth of unicellular eukaryotic organisms (e.g., saccharomyces cerevisiae), the cell culture medium being selected from yeast extract peptone glucose (YPD), yeast extract peptone glycerol (YPG) and yeast extract peptone glucose (YPAD) culture medium.

[0247] According to some embodiments, the cell culture medium is suitable for the growth of prokaryotes, for example the bacteria is E. coli or probiotics, such as bacteria of the genera Bifidobacterium and Lactobacillus. According to one embodiment, the method comprises adding the stabilized ACC to a medium suitable for bacterial growth, such as LB or M9.

[0248] According to any of the above embodiments, stable ACC is added to the cell culture medium according to any of the above embodiments at a final concentration of about 0.1 to about 20 mM, about 0.5 to about 15 mM, about 1 to about 10 mM, about 2 to about 8 mM, about 3 mM to about 6 mM, or about 4 mM to about 5 mM. In more specific embodiments, stable ACC is present at a concentration of 0.5 to about 4 mM, about 1 to about 3 mM, about 1.5 to about 2.5, or about 1, 1.5, 2, or 2.5 mM.

[0249] According to one embodiment, ACC is formulated in solid, liquid or semi-liquid form. In a specific embodiment, the stable ACC is added in the form of a suspension. In yet another embodiment, the suspension is a freshly prepared suspension.

[0250] According to any of the above embodiments, ACC is stabilized by at least one stabilizer defined above. In certain embodiments, the ACC stabilizer is independently an organic acid at each occurrence; a phosphorylated, phosphonated, sulfated or sulfonated organic compound; a phosphate or sulfate of a hydroxycarboxylic acid; an organic amine compound; an organic compound containing a hydroxyl group; an organic phosphorus compound or a salt thereof; phosphorylated amino acids and their derivatives, bisphosphonates; an organic phosphate compound; an organic phosphonate compound; an organic polyphosphate, an inorganic polyphosphate, an inorganic phosphorous acid, an organic compound having a plurality of functional groups as defined above; an inorganic phosphate and polyphosphate compound; an organic compound having a polyphosphate chain; an organic surfactant; an essential inorganic ion; a carbohydrate and its derivatives, a protein, a phosphorylated protein, a natural and synthetic biopolymer and its derivatives or any combination thereof. According to another embodiment, the stabilizer is selected from the group consisting of phosphoserine, adenosine triphosphate, adenosine diphosphate, phytic acid, citric acid, etidronic acid, pyrophosphate, polyphosphate, triphosphate, ethanol, hexametaphosphate, chitin and any combination thereof. These compounds are as defined above. According to some embodiments, ACC is stabilized by more than one stabilizer, for example by 2 or 3 stabilizers.

[0251] According to some embodiments, the stabilizer is selected from the group consisting of polyphosphates such as triphosphates, phosphorylated amino acids such as phosphoserine, diphosphates, citric acid, and any combination thereof such as a combination of triphosphate and citric acid or a combination of phosphoserine and citric acid.

[0252] According to another aspect, the present invention provides a method for preparing a cell culture medium comprising amorphous calcium carbonate (ACC) stabilized by at least one stabilizer, the method comprising adding stabilized ACC to the cell culture medium.

[0253] According to certain aspects, the present invention provides a kit comprising amorphous calcium carbonate (ACC) stabilized by at least one stabilizer and instructions for use of the ACC in combination with a cell culture medium. According to one embodiment, the stabilized ACC is ACC for use as a cell culture medium supplement. According to one embodiment, the present invention provides a kit comprising calcium chloride, sodium carbonate, at least one stabilizer and instructions for preparing stable ACC from the calcium chloride, sodium carbonate, at least one stabilizer and instructions for use of the stable ACC in combination with a cell culture medium. According to one embodiment, the calcium chloride, sodium carbonate and / or at least one stabilizer are present as an aqueous solution.

[0254] According to another embodiment, the kit comprises a cell culture medium supplement comprising ACC stabilized by at least one stabilizer and instructions for use of the supplement in combination with a cell culture medium. According to one embodiment, the cell culture medium supplement consists of ACC stabilized by at least one stabilizer.

[0255] According to any of the above embodiments, the kit further comprises a cell culture medium as defined above. Thus, in one embodiment, the kit comprises ACC stabilized by at least one stabilizer, a cell culture medium, and instructions for use. In other embodiments, the kit comprises a cell culture medium supplement containing ACC stabilized by at least one stabilizer, a culture medium, and instructions for use.

[0256] The ACC stabilized by at least one stabilizer and the cell culture medium are as described above.

[0257] According to some embodiments, the stabilizer is selected from the group consisting of polyphosphates such as triphosphates, phosphorylated amino acids such as phosphoserine, diphosphates, citric acid, and any combination thereof such as a combination of triphosphate and citric acid or a combination of phosphoserine and citric acid.

[0258] According to some embodiments, the ACC composition comprises a combination of stabilizers disclosed above.

[0259] According to any one of the above-mentioned embodiments, stable ACC can be formulated into solid, liquid or semi-liquid preparations. Therefore, in one embodiment, the stable ACC used as a supplement for cell culture medium is formulated into solid, liquid or semi-liquid. According to some embodiments, stable ACC is formulated into solid forms, such as powders, tablets, capsules or particles. Therefore, in a specific embodiment, the stable ACC used as a supplement for cell culture medium is formulated into powder. According to other embodiments, the stable ACC used as a supplement is formulated into liquid or semi-liquid. According to one embodiment, the liquid form is a suspension or an emulsion, and the semi-liquid form is a gel or a colloid. In a specific embodiment, stable ACC is formulated into a suspension. Therefore, in one embodiment, before using the culture medium, the stable ACC formulated into a powder or a suspension is added to the cell culture medium. According to an even more specific embodiment, stable ACC is added as a freshly prepared suspension.

[0260] The kit of the present invention may also include a cell culture medium. Cell culture medium is well known in the art, and any culture medium may be used. In some embodiments, the cell culture medium is suitable for growing biological cultures, the culture being selected from the culture of cells, tissue cultures, organ cultures or organs. The culture of cells may be a culture of eukaryotic or prokaryotic cells. Specifically, the cell culture medium refers to a culture medium suitable for eukaryotic cell cultures, tissue cultures or organ growth. In some specific embodiments, the culture medium may be a complete culture medium, a basal culture medium, a basal culture medium supplemented with a cell culture medium supplement, a culture medium with various amounts of serum or a chemically defined culture medium.

[0261] According to some embodiments, the cell culture medium is selected from DMEM, RPMI 1640, MEM, IMDM, L-15 medium (Leibovitz), MCDB medium, medium 199, opti-MEM and DMEM / F-12, Schneider fruit fly medium, Grace insect medium, IPL-41 insect medium Sf-900, serum-free insect medium, Shields and Sang M3 insect medium, TC-100 insect medium, TNM-FH insect medium, Ham's F-12, Ham's F-10, GMEM, Ames medium, Eagle's basic medium (BME), Claycomb, Click medium, Glasgow's minimum essential medium (GMEM), MegaCell medium, McCoy's 5A modified medium, NCTC medium, Williams medium E, Waymouth medium, TC-10 and IPL-10 medium. In a specific embodiment, the cell culture medium is selected from a monoculture medium, such as Quinn's Advantage TM (SAGE) Medium 1-Step TM , continuous culture media, such as Quinn's Advantage TM Cleavage medium, MEDICULT, universal IVM, DMEM, RPMI 1640, MEM, IMDM, opti-MEM, GMEM, Ham's F-12, DMEM / F-12, Schneider Drosophila medium, Grace insect medium, Sf-900, TC-10, IPL-10, B5, N6 or Nitsch medium. Another example of a culture medium is a culture medium for sperm isolation, such as PureCeption TM 、Multipurpose Handling Quinns TMSperm washing medium, MultipurposeHandling and modified HTF medium-HEPES with gentamicin. According to other embodiments, the culture medium is a medium for oocyte maturation, such as SAGE TM In vitro maturation medium (IVM) and BO-IVM oocyte maturation medium. According to further embodiments, the cell culture medium is selected from a medium for fertilization, a medium for embryo development, and a medium for embryo or / and gamete maturation, processing and / or cryopreservation.

[0262] According to some embodiments, the cell culture medium is suitable for yeast growth, and the cell culture medium is selected from the group consisting of YPD, YPG and YPAD.

[0263] According to some embodiments, the cell culture medium is suitable for the growth of prokaryotes, said medium being selected from the group consisting of LB and M9.

[0264] According to one embodiment, the present invention provides a kit comprising calcium chloride, sodium carbonate, at least one stabilizer, and instructions for preparing stable ACC and instructions for using the ACC in combination with a cell culture medium. According to some embodiments, the calcium chloride and sodium carbonate are present as aqueous solutions. According to one embodiment, the at least one stabilizer is present as one or two separate solutions. According to one embodiment, the instructions for preparing ACC include instructions for mixing a solution comprising calcium chloride and at least one stabilizer with a sodium carbonate solution and further adding a solution comprising at least one stabilizer.

[0265] According to some embodiments of the present invention, the terms "capable of promoting" and "promoting" are used interchangeably in some embodiments.

[0266] In any of the above embodiments, the term "comprising" includes the meaning of "consisting of", and can be replaced by it.

[0267] As used herein, the term "about" when referring to a measurable value such as an amount, duration of time, etc., is meant to encompass variations of + / - 10%, or + / - 5%, or + / - 1%, or even + / - 0.1% from the specified value.

[0268] Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention. Example

[0269] Example 1. Effect of ACC on SC-DRG co-cultures

[0270] method

[0271] Culture medium

[0272] The culture medium included: 90% Dulbecco's modified eagle medium-nutrient mixture F-12 (DMEM-F12) decalcified (medium without calcium ions, specific preparation), 10% heat-inactivated fetal bovine serum (FBS), 6 g / L D-glucose, 2 mM glutamine, 25 μg / mL gentamicin and 0.05 ng / mL insulin-like growth factor 1 (IGF-I) (all purchased from Biological-Industries, Israel).

[0273] NVR-Gel as a substrate for neuronal culture

[0274] NVR-Gel (NVR Labs) consists of two main components: high molecular weight hyaluronic acid (HA, 3x10 6 Da, BTG, Israel) and laminin (Sigma). For neuronal cell culture, 1% NVR-Gel was diluted with culture medium to a final concentration of 0.3-0.5%. The gel has the texture of a viscous liquid, which easily and successfully adheres embedded cells or isolated fragments to plastic or glass substrates and enables the outgrowth of neural fibers in a 3D pattern.

[0275] Preparation of neuronal tissue cultures

[0276] All experiments were authorized and conducted by the local ethics committee on animal experiments recognized by the Israeli authorities. Fixed organotypic cultures of dorsal root ganglia (DRG) and spinal cord (SC) and cultures of dissociated brain cells were prepared from rat fetuses (gestational day 15, Lewis inbred strain, Harran, Israel). After dissection, the separated tissue was immediately cut into small slices (400 μm thick) with a Macwain tissue chopper. In these studies, two tissue culture strategies were used. In the first method, tissue slices were directly inoculated into 12-well culture plates containing 1 mL of culture medium containing 0.3%-0.5% NVR-Gel. In the second method, tissue slices were further dissociated with trypsin-EDTA solution for 30 minutes and washed with culture medium. Subsequently, the dissociated cells were added to a suspension of chitosan powder or gastrolith powder (microcarrier, MC) and suspended and incubated at 37°C for 4 days. The formed floating cell / MC aggregates were then collected and seeded in 12-well culture plates containing 1 mL of culture medium containing 0.3%-0.5% NVR-Gel.

[0277] Ca 2+ Supplement Sources

[0278] During the seeding phase, Ca was added to the gel once to a final concentration of 1 or 2 mM. 2+ Sources (listed below) were added to the nutrient medium at each successive feeding. Calcium sources were as follows: ACC-etidronic acid (ACC-ET) (fresh suspension); ACC-phosphoserine (ACC-PS) (fresh suspension); gastrolith (dissolved with 0.1 M HCl and then neutralized with 1 M NaOH); gastrolith powder; CCC-aqueous suspension of crystalline calcium carbonate (commercial nanoparticle powder); and CaCl2 solution-control.

[0279] Beginning 24 hours after setting up the cultures, the cultures were monitored daily by phase contrast microscopy.

[0280] The suspension of fresh ACC preparation is composed of particles that form a stable suspension. Gastric stone is the natural ACC isolated from crabs and can only be purchased as a dry powder. In this form, cells cannot obtain its other characteristic components (such as calcium ions and proteins). In order to improve their bioavailability, gastric stone powder is dissolved in 0.1M HCl (it simulates the acidity present in the stomach) and then neutralized with 1M NaOH.

[0281] Immunofluorescence staining of neuronal cultures

[0282] After removing the culture medium, the dorsal root ganglion (DRG) cultures were washed with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 minutes and then washed again with PBS. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS and then immunoblocked (to avoid nonspecific staining) with 1% bovine serum albumin (BSA) in PBS at room temperature for 1 hour. The samples were then incubated with rabbit anti-neurofilament antibodies (NF, Novus Biologicals, 1:500) to visualize neurite outgrowth. The primary antibody was diluted in 0.1% BSA and 0.05% Tween-20 (diluent buffer) in PBS and incubated overnight with the samples at 4°C. After washing with 0.05% Tween-20 in PBS (washing buffer), DRG samples were incubated with secondary antibody Alexa-Fluor-594-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, USA, 1:800 in diluent buffer) at room temperature for 1 hour. Finally, the samples were rinsed again with washing buffer and mounted with mounting medium (Immco Diagnostic, USA). All images were observed with an Olympus IX70 microscope.

[0283] result

[0284] The effect of various calcium preparations was examined in SC-DRG co-cultures. In general, cultures contained 400 microns of SC slices, together with attached or detached DRG slices. It can be considered that, compared with CCC and CaCl2, all examined ACC preparations (ACC-ET, ACC-PS and gastric stone) significantly promoted neuronal fiber regeneration. Table 1 shows the effect of various calcium preparations (2mM Ca 2+ The proportion of explants (out of 6) showing neurofibrillary sprouting after 4 days of culture in the presence of ACC-ET (100% of explants), followed by cultures exposed to ACC-PS and gastroliths (66.6% of explants). CCC and CaCl2 induced neuronal sprouting only from 50% of the explants, and the stabilizers alone induced even lower percentages (0-33%). During culture establishment (after the first week of culture), the sprouting of neurons was mainly observed in cultures exposed to various ACC preparations ( Figure 1 ), the regenerated nerve fibers become longer, thicker and branch out until a neuronal network is formed.

[0285] Table 1: Effects of ACC preparation on early neurofibril sprouting from SC-DRG co-cultures.

[0286] Types of calcium preparations Cultures with axon regeneration (%) ACC-ET (fresh suspension) 100 ACC-PS (fresh suspension) 66.6 Gastric Stone 66.6 CCC (Crystallized Calcium Carbonate) 50 <![CDATA[CaCl2 aqueous solution (control)]]> 50 ET 33.3 PS 0

[0287] Example 2. Effects of ACC on brain cultures

[0288] The effect of ACC on brain cell-MC aggregate cultures seeded in gel after 4 days in suspension was studied (see Methods section of Example 1). Figure 2 ACC promotes nerve fiber regeneration more significantly than calcium chloride. This is particularly significant when comparing the number and length of nerve fibers between the two treatments.

[0289] Example 3 - Effects of ACC preparation on healthy skeletal muscle cells

[0290] method

[0291] Preparation of skeletal muscle cultures

[0292] Fixed skeletal muscle cultures were prepared from healthy 1-day-old neonatal rats (Sprague Dawley, Harran, Israel). Muscle tissue was dissected from the hind legs and minced thoroughly. Digestion was performed with trypsin-EDTA while gently grinding. After 30 minutes, the supernatant containing the dissociated cells was collected and fresh trypsin-EDTA was added. This procedure was repeated two more times. All supernatants were then pooled, centrifuged, and the cell pellets were resuspended in proliferation medium. Cells were seeded in gelatin-coated 12-well culture plates containing 1 mL of proliferation medium at 1 × 10 5 Cells / well. Two days later, the culture medium was changed to fusion medium, which was then changed twice a week. Beginning 24 hours after setting up the culture, the culture was monitored by daily phase contrast microscopy. On a predetermined date, the culture was fixed in methanol for 20 minutes and then stained with Giemsa to assess the number of myotubes formed in the culture over time.

[0293] Gelatin-coated culture plates

[0294] A 1% porcine gelatin stock solution in water was sterilized by autoclave. Once cooled, 500 μl of the solution was added to each well of a 12-well culture plate. After incubation at room temperature for 20 minutes, excess solution was removed and cells were seeded.

[0295] Proliferation medium

[0296] For the proliferation phase, cells were cultured in DMEM / F12 (containing 1 mM Ca 2+ ) + 10% FBS, 25 μg / mL gentamicin and 2 mM L-glutamine.

[0297] Fusion medium

[0298] For the fusion stage, the proliferation medium was replaced with fusion medium, which was prepared as follows: DMEM / F12 (containing 1 mM Ca 2+ ), 2% horse serum (HS), 2 mM L-glutamine, 25 μg / mL gentamicin, and 4 units / 100 mL insulin (all purchased from Biological-Industries, Israel).

[0299] List of calcium preparations tested

[0300] Use the Ca listed in Table 2 2+ The fusion medium was enriched with various calcium preparations at a concentration of 1 mM (since the medium already contained 1 mM calcium ions, Ca 2+The final concentration was 2 mM). Control cultures were grown without further calcium addition or cultures were enriched with free (soluble) stabilizer. The experiment was blinded by labeling the various calcium preparations with just an arbitrary number.

[0301] The components were added in one of the following ways: (i) aqueous suspension of dry material; (ii) aqueous suspension of fresh material (before drying), or (iii) dissolved in HCl (to simulate the acidity present in the stomach. After dissolution was complete, the resulting solution was neutralized with 1 M NaOH).

[0302] Table 2 - List of materials tested

[0303]

[0304] result

[0305] Effects of dry ACC on healthy skeletal muscle

[0306] In the first stage, dry ACC powder was used. The powder (the stabilizers used in its formulation are listed in Table 2) was suspended in water and then added to the culture medium at a concentration of 1 mM. Since the culture medium already contained 1 mM calcium ions, Ca 2+ The final concentration was 2 mM. Some results are shown in Figure 3 (left hand side), it is revealed that cultures exposed to ACC already exhibited the early formation of numerous myotubes within 4 days of culture, with no significant differences between the different ACC preparations. In control cultures exposed to added CCC or CaCl2, myotube formation was observed later. After 7 days, cultures treated with ACC exhibited a large number of long and thick myofibers, whereas fewer, thinner and shorter myofibers were formed in cultures treated with CCC and CaCl2 ( Figure 3 ; right-hand side).

[0307] It should also be noted that in cultures exposed to ACC preparations, muscle contractions were already observed on day 7 after inoculation, whereas in cultures exposed to added CCC or CaCl2, muscle contractions appeared only after 10 days or more.

[0308] From the above in vitro results, it can be concluded that all ACC preparations promoted myotube formation and early muscle contractility in healthy striated muscle cultures.

[0309] Example 4 - Evaluation of the Effect of ACC on Duchenne Muscular Dystrophy Muscular Cell Line - In Vitro Studies

[0310] method

[0311] Mdx cell preparation

[0312] The effects of different calcium preparations on the mdx cell line (a model of Duchenne muscular dystrophy), kindly provided by Prof. (Emeritus) David Yaffe, Weizmann Institute of Science, Israel, were investigated.

[0313] Cells from the Mdx cell line were seeded in gelatin-coated 12-well culture plates containing 1 mL of proliferation medium at 3 × 10 4 cells / well. Two days later (~66% confluence), the medium was changed to fusion medium, which was then changed twice a week. Various calcium preparations were added to the fusion medium individually according to the treatments described in Table 3. 2+ The culture was enriched with various calcium preparations at a concentration of 1 mM. Since the culture medium already contained 1 mM calcium ions, Ca 2+ The final concentration is 2 mM.

[0314] Table 3: Calcium sources

[0315] deal with Calcium source (2 mM) Comparison <![CDATA[CaCl2]]> ACC-ET Amorphous calcium carbonate-etidronic acid ACC-PS Amorphous calcium carbonate-phosphoserine

[0316] The effects of the tested calcium preparations on cell proliferation, fusion to form myotubes, and muscle contraction were monitored by daily phase contrast microscopy. On predetermined days, cultures were fixed in methanol for 20 minutes and then stained with Giemsa to assess the number of myotubes formed in the cultures over time.

[0317] Creatine Kinase (CK) Assay in Muscle Tissue Cultures

[0318] In muscle cell cultures, CK is an indicator of myotube formation, and CK levels increase in direct correlation with the progression of myotube formation in muscle culture (in tissue culture plates). On predetermined days, cells were harvested from culture wells (using a rubber policeman) and stored in 1 mL PBS (without Ca) at -70°C. 2+ ) until analyzed. For CK measurement, cell samples were thawed and physically lysed using a sonicator to release CK from muscle myotubes. CK concentration was determined using a creatine kinase activity assay kit (CK-NAC REAGENT SET, CURTISS, CHEM-INDEX INC, Hialeah, FL, USA).

[0319] result

[0320] The results of the above experiments are presented in Figure 4 and Figure 5It can be clearly seen that the addition of ACC promotes cell fusion and myotube formation better than the addition of a conventional calcium ion source (CaCl2). This surprising result was demonstrated in both biochemical (CK activity) and morphological (microscopy) analyses ( Figure 4 and Figure 5 ). In addition, in cultures exposed to ACC preparations, muscle contractions were already observed on day 7 after inoculation, whereas in controls, muscle contractions appeared only after 10 days or more. Therefore, it can be concluded that ACC supplementation has the potential to treat DMD patients.

[0321] Example 5 - Effect of calcium sources on primary mdx mouse cells

[0322] method

[0323] Extraction of primary cells

[0324] Under sterile conditions, thigh muscles were removed from the hind legs of newborn (one-day-old) mdx mice and washed in PBS to remove excess blood cells. The muscles were minced into small pieces. For enzymatic decomposition, the muscle fragments were placed in a beaker containing trypsin-EDTA solution (0.25mM). To ensure cell separation, the mixture was placed on a blender and stirred gently for 20 minutes at room temperature. The soup was collected and centrifuged at 300×g for 5 minutes. The precipitate was resuspended in DMEM containing FBS. The trypsinization step was repeated for 3 more cycles. All supernatants were combined into a test tube. Visually determine cell separation (using phase contrast microscopy). Cell density was determined using a hemocytometer.

[0325] The cells were plated at 2x10 5 The cells were plated at a concentration of 10 cells / well in a 12-well plate. The culture medium used was DMEM / F12W / O Ca supplemented with 15% FBS and 2 mM L-glutamine and gentamicin (25 μg / ml). 2+ Calcium was added to the culture medium individually according to the treatments as described in Table 4. On day 2 (~66% confluence), the culture medium was changed to fusion medium (DMEM / F12 without Ca 2+ , supplemented with 10% HS, insulin (4 units / 100 ml) and gentamicin (25 μg / ml). The medium was changed every 3 days.

[0326] Table 4. Calcium sources used in primary cell studies

[0327] deal with Calcium source (2 mM) Comparison <![CDATA[CaCl2]]> ACC-PP Amorphous calcium carbonate stabilized by polyphosphate ACC-PS Amorphous calcium carbonate stabilized by phosphoserine

[0328] Cell proliferation and confluence were qualitatively monitored daily. Cultures were fixed on days 2, 3, 4, 5 and 7 and stained with Giemsa or with myosin antibodies.

[0329] result

[0330] The results are presented in Figure 6 and Figure 7 The beneficial effects of the ACC preparation were demonstrated by myotube formation, especially at early time points, day 3 and day 4, compared to the control. On days 5 and 7, the differences in the formed myotubes became indistinguishable. There was a high correlation between Giemsa staining and myosin staining.

[0331] Promotion of embryonic development in ACC-supplemented medium

[0332] Example 6.

[0333] Materials and methods:

[0334] CBA male mice were mated with BL C57 female mice. The mice were maintained on a 12-hour light cycle schedule with unlimited water and food supply. Approximately 6 to 8 weeks after receiving the offspring, each female mouse was injected IP with 5 IU pregnant mare serum gonadotropin (PMSG).

[0335] After 48 hours, each female mouse was injected with 5IU human chorionic gonadotropin (hCG). Then the male mice that will be proved to be fertile were put together with the superovulated female mice. The next morning, the female mice were checked for copulatory plugs. After 24 hours (about 36 hours after mating), the females with copulatory plugs were euthanized, and the embryos of the 2-cell stage were taken out from the oviduct as follows: the oviduct was dissected in Quinn's Advantage cleavage medium (SAGE, Origio, Denmark), and the embryos were transferred to 20 μL droplets with Quinn's Advantage cleavage medium (SAGE, Origio, Denmark) covered with mineral oil, and cultivated at 37.0°C at 5% CO2 and atmospheric oxygen. The collected embryos continue to grow, i.e., in vitro culture (IVC), until the blastocyst / hatching stage. In order to test the effect of amorphous calcium carbonate, Quinn's Advantage cleavage medium (hereinafter "cleavage medium") is supplemented with different additives, i.e. amorphous calcium carbonate stabilized by polyphosphate (ACC-PP), crystalline calcium carbonate (CCC), polyphosphate (PP), phosphoserine (PS) or sodium carbonate (NaCO3). All supplements are added as freshly prepared suspensions; the preparation of ACC supplement is carried out under sterile conditions. Untreated cleavage medium (without any additives) is used as a control (identified as Cont Q). Embryos are evaluated by microscopic observation every day in order to detect the following stages: 2 cells, compaction stage, blastocyst formation and hatching stage. The proportion (percentage) of embryos arriving at each stage is calculated (the number of 2 cell embryos is set to 100%).

[0336] result

[0337] The embryonic development in the cleavage medium supplemented with 1.7 mM amorphous calcium carbonate stabilized with PP (ACC-PP) was compared with the control (development in the untreated medium of Cont Q-). The embryonic number at each developmental stage and their proportion in the initial embryonic number (the latter is presented in brackets) are presented in Table 5.

[0338] Table 5. Comparison of embryo growth in untreated cleavage medium and medium supplemented with ACC-PP

[0339] 2 cells / d1 Densification / d2 Blastocyst / d3 Hatching / d4 Cont Q 12 2(16.6) 10(83.3) 8(66.6) ACC-PP 40 17(42.5) 40(100) 32(80)

[0340] Example 7.

[0341] The embryonic development in cleavage medium supplemented with 1.7 mM AC-PP or calcium chloride (CaCl2) was tested in two separate tests (A and B). In test A, embryos were grown until the blastocyst stage, and in test B, embryos were grown until the hatching stage. The number of embryos at each developmental stage and their proportions (in brackets) to the initial number of embryos are presented in Table 6 (Test A) and Table 7 (Test B).

[0342] Table 6: Test A: Comparison of embryos grown in cleavage medium supplemented with 1.7 mM CaCl2 or ACC-PP

[0343] 2 cells / d1 Densification / d2 Blastocyst / d3 <![CDATA[CaCl2]]> 25 16(64) 16(64) ACC-PP 26 24(92.3) 22(84.6)

[0344] Table 7: Test B: Comparison of embryos grown in cleavage medium supplemented with 1.7 mM CaCl2 or ACC-PP

[0345] 2 cells / d1 Densification / d2 Blastocyst / d3 Hatching / d4 <![CDATA[CaCl2]]> 27 20(70) 20(74) 11(40.7) ACC 23 23(100) 23(100) 23(100)

[0346] Example 8.

[0347] Embryonic development in cleavage medium supplemented with 1.7 mM CaCl2, 1.7 mM ACC-PP or 0.85 mM ACC-PP (half the initial concentration of ACC-PP, identified as ACC-PP0.85). The number of embryos at each developmental stage and their proportion in the initial number of embryos (in brackets) are presented in Table 8.

[0348] Table 8. Comparison of embryos grown in cleavage medium supplemented with 1.7 mM CaCl2, 1.7 mM or 0.85 mM AC-PP.

[0349] 2 cells / d1 Densification / d2 Blastocyst / d3 Hatching / d4 <![CDATA[CaCl2]]> 10 3(30) 8(80) 3(30) ACC 11 7(63.6) 9(81.8) 7(63.6) ACC-PP0.85 11 8(72.7) 9(81.8) 8(72.7)

[0350] Example 9.

[0351] The embryonic development in the cleavage medium supplemented with 1.7 mM CaCl , 1.7 mM or 0.85 mM ACCC-PP (ACC-PP0.85) was tested and compared with the development in the untreated medium. The embryonic number at each developmental stage and their ratio (in brackets) in the initial embryonic number are presented in Table 9.

[0352] Table 9. Comparison of embryos grown in untreated cleavage medium and cleavage medium supplemented with 1.7 mM CaCl2, 1.7 mM or 0.85 mM AC-PP.

[0353] 2 cells / d1 Densification / d2 Blastocyst / d3 Hatching / d4 Cont Q 42 30(71.4) 38(90.4) 15(35.7) <![CDATA[CaCl2]]> 30 16(53.3) 24(80) 13(43.3) ACC-PP 30 23(76.6) 27(90) 12(40) ACC-PP0.85 30 21(70) 30(100) 24(80)

[0354] Example 10

[0355] In this experiment, 4 female mice of 5 months age are euthanized.As described in the materials and methods, the embryos from each mouse are collected, but embryos are not brought together.Embryos from each oviduct of each mouse are supplemented with 1.7mM CaCl or 1.7mMACC-PP cleavage medium, grown separately in culture.In this way, we can also assess the difference between the mice, because the embryos from each female mouse are siblings.The embryo number at each developmental stage and their ratio (in brackets) in the initial embryo number are presented in Table 10.

[0356] Table 10. Comparison between embryos from 4 female mice grown separately in cleavage medium supplemented with 1.7 mM CaCl2 or ACC-PP.

[0357]

[0358] Example 11.

[0359] In this experiment, 7 female mice of 6 months age were euthanized. As described in Materials and Methods, embryos were collected from each mouse, but the embryos collected from No. 1 and No. 2 mice were not collected together, but grown separately as in Example 5. The embryo from No. 1 mouse was grown in untreated cleavage medium or the medium (ACC-PP2.6) supplemented with 2.6mM AC-PP. The embryo from No. 2 mice was grown in the cleavage medium (identified as ACC-PP 1.3 and ACC-PP 0.6, respectively) supplemented with 1.3mM or 0.6mM ACC-PP. The embryos from No. 3-7 mice were all collected together, and grown with 1.6mM polyphosphate (PP). In addition, the diameter and volume of blastocysts were calculated. The embryo number at each developmental stage and their ratio (in brackets) in the initial embryo number are presented in Table 11.

[0360] Table 11: Comparison of sibling embryos grown in untreated cleavage medium or in medium supplemented with different concentrations of ACC-PP. Pooled embryos from mice 3-7 were grown in medium supplemented with polyphosphate.

[0361]

[0362]

[0363] It was found that hatched blastocysts grown with 2.6 mM ACC-PP were 28% larger in diameter and twice as large in volume as controls.

[0364] Example 12.

[0365] In this experiment, 7 female mice of 6 months age were euthanized. The embryos from each mouse were collected as described in Materials and Methods (Example 6). Embryos were pooled together and divided into 5 groups, grown in untreated cleavage culture medium (ContQ), or grown in the culture medium supplemented with 2.6mM, 1.3mM, 0.6mM AC-PP (respectively labeled as ACC-PP 2.6, ACC-PP1.3 and ACC-PP 0.6) or 1.6mM polyphosphate (PP). The embryo number at each developmental stage and their ratio (in brackets) in the initial embryo number are presented in Table 12.

[0366] Table 12: Comparison of embryos grown in untreated cleavage medium or medium supplemented with 2.6 mM, 1.3 mM or 0.6 mM ACC-PP or with 1.6 mM PP.

[0367]

[0368] Example 13.

[0369] In this experiment, 6 female mice of 6 months age were euthanized. The embryos from each mouse were collected as described in the materials and methods. Embryos were pooled together and divided into 3 groups: one group was used as a control (Cont Q), and in another two groups, embryos were grown in the cleavage culture medium supplemented with 3.3mM ACCC-PP (ACC-PP3.3) or 3.3mM commercially available nano-crystalline calcium carbonate (CCC). All three culture mediums were prepared under aseptic conditions. The embryo number at each developmental stage and their ratio (in brackets) in the initial embryo number are presented in Table 13.

[0370] Table 13. Comparison of embryos grown in untreated cleavage medium versus medium supplemented with 3.3 mM ACC-PP or 3.3 mM CCC.

[0371]

[0372] Example 14.

[0373] In this experiment, 6 female mice of 7 months age were euthanized.According to the embryo collected from each mouse described in Materials and Methods (Example 6).Embryos are brought together and grown in the cleavage medium of the phosphoserine (PS) of 1.7mM CaCl , 1.7mMACC-PP, 0.8mMACC-PP (ACC-PP0.8) or 1.7mM.Embryo numbers at each developmental stage and their ratio (in brackets) in initial embryo numbers are presented in Table 14.

[0374] Table 14. Comparison of embryos grown in cleavage medium supplemented with 1.7 mM CaCl2, 1.7 mM AC-PP, 0.8 mM AC-PP and 1.7 mM PS.

[0375]

[0376] It can be seen that in this particular experiment, the addition of calcium chloride had a negative impact on embryo development, resulting in lower blastocysts and percentage of hatched blastocysts.

[0377] Example 15.

[0378] In this experiment, 6 female mice of 6 months age are euthanized.As described in materials and methods (embodiment 6), the embryo from each mouse is collected, but the embryo from No. 1 and No. 2 mice is not brought together, but grown separately as sibling experiment (referring to embodiment 10).Grow in untreated cleavage culture medium or in the culture medium supplemented with 1.7mM nanocrystalline calcium carbonate (CCC) from No. 1 female embryo.Grow in the culture medium supplemented with 1.7mMACC-PP or 1.7mM sodium carbonate (NaCO 3) from No. 2 mice.Grow in all embryos from No. 3-6 mice, and in untreated culture medium or in the culture medium supplemented with 1.7mM NaCO 3 or 1.7mM ACC-PP.In addition, calculate the diameter and volume of blastocyst (referring to table 15).

[0379] Table 15: Developmental assessment of sibling embryos and pooled embryos from two mice grown in differently supplemented cleavage media.

[0380]

[0381] It can be seen that the addition of sodium carbonate resulted in very poor embryonic development compared to the control or the addition of ACC-PP. It was also found that hatched embryos grown in cleavage medium supplemented with 2.6 mM ACC-PP had a diameter 28% larger than the control and a volume that was twice as large as the control.

[0382] Example 16. Preparation of 10% TP-1% citrate ACC (ACC stabilized with 10% triphosphate and 1% citric acid) formulated as a cell culture medium supplement.

[0383] 36 ml of 3% calcium chloride solution was mixed with 4 ml of 0.27% citric acid solution and 10 ml of 0.5406% triphosphate solution. Then 40 ml of 1.9485% sodium carbonate solution was added to precipitate ACC. 10 ml of stabilizing solution containing 0.5406% triphosphate was added to the ACC suspension to produce a stable ACC suspension. The resulting suspension was used as Quinn'sTM Cleavage medium or Quinn's TM Supplement for 1-Step medium. Add the suspension to a final concentration of ACC of 1.7 or 3.4 mM. Alternatively, filter the suspension with a Buchner funnel, wash the filter cake with water, and further dry the filter cake, for example, in an oven. Add the powder to the cleavage medium to a final concentration of 1.7 or 3.4 mM.

[0384] Embodiment 17

[0385] Four female mice aged 6-8 weeks were euthanized in this experiment. Embryos from each mouse were collected as described in Materials and Methods. Stable ACC (ACC stabilized with 10% triphosphate and 1% citric acid) was prepared as described in Example 16. Embryos from each female were isolated and a portion was grown on untreated SAGE 1-Step TM The second part was grown in SAGE 1-Step medium supplemented with 1.7mM AC-PP or 3.4mM AC-PP. TM Grown in culture medium. Figure 8 It can be seen that the addition of stable ACC has a positive effect on embryo development, resulting in higher blastocysts and the percentage of hatched blastocysts, especially in the case of 3.4mMACC. It was surprisingly found that embryos grown in cleavage medium supplemented with stable ACC showed rapid cleavage and a higher hatching rate.

[0386] Example 18. Preparation of ACC formulated as a cell culture medium supplement.

[0387] Compositions of ACC stabilized by different stabilizers (triphosphate (TP), hexametaphosphate (HMP), pyrophosphate (Pyr), phosphoserine (PS), etidronic acid (ET), zoledronic acid (ZA), or methylenephosphonic acid (MA)) were prepared. In a typical procedure, a calcium solution (300 ml of water, 24 g of calcium chloride and stabilizer) and a carbonate solution (200 ml of water and 17.3 g of sodium carbonate) were mixed together to precipitate ACC. A stabilizer solution (100 ml of water and stabilizer; the content of stabilizer in the calcium and stabilizer solutions is presented in Table 16) was added to the ACC suspension to produce a stable ACC suspension. The suspension can be used as a supplement. The ACC was then filtered using a Buchner funnel, and the filter cake was washed with water. The suspension was dried to obtain a powder. The powder can be added as a cell culture medium supplement, or resuspended in an aqueous medium and added as a suspension.

[0388] Table 16. Stabilizer content in different ACC compositions

[0389]

[0390] In other embodiments, the composition is prepared as described above, and citric acid is added to the calcium solution to obtain 1% citric acid in the final composition. The suspension itself is used as a culture medium supplement. Alternatively, the suspension is further washed with water and dried to obtain a powder. The powder can be added to any cell culture medium or resuspended in an aqueous culture medium.

[0391] Example 19. Growth of MBA13 stem cells (bone marrow stromal cells) into osteoblasts

[0392] Materials and methods

[0393] Two days after thawing, MBA-13 cells (obtained from Weizmann Institute of Science, Prof. Dov Zipori) were resuspended in recombinant trypsin solution and cultured at 1×10 4 The cells were seeded in a 96-well plate at a concentration of 10 cells / well (day "0"), and MSCs were supplemented with a mesenchymal stem cell (MSC) supplement mixed medium (Biological Industries cat#05-201-06) at a ratio of 50 ml:300 μl. XF basal medium (Biological Industries, cat#05-200-1A). 2+ Mg 2+ ) at a ratio of 1:100 pre-coated with MCS attachment solution for cell seeding. Rows AH of columns 5-8 of a 96-well plate were pre-coated with gelatin 0.1% for 30 minutes at room temperature.

[0394] On day 2, when more than 80% cell confluence was reached (~48h), the medium was changed to MSCgo Rapid Osteogenic Medium (cat#05-442-1B) containing factors that promote osteoblast differentiation. After the medium change, rows AC were supplemented with an additional 1mM calcium (total 2.488mM calcium) from amorphous calcium carbonate (ACC) stabilized with 10% triphosphate + 1% citric acid; rows DF were supplemented with an additional 1mM calcium from calcium chloride (total 2.488mM calcium); rows G in the plate were treated with MSCgo medium (total 1.488mM calcium). Row H of the plate was treated with XF Nutrient Base Medium + Supplement Mix (1.488 mM calcium total).

[0395] On day 4, the culture medium was exchanged with a fresh preparation of ACC.

[0396] In parallel, control plates were also seeded with MDX cell lines derived from injured muscle of MDX mice. Staining of these cells was used to set background staining of cell intrinsic calcium and also to eliminate the possibility of ACC-treated calcium deposits being stained. Prior to seeding, wells were coated with gelatin, which serves as a standard matrix for MDX cell attachment. MDX cells were seeded at 3 × 10 4 The cells were seeded at a concentration of 10 cells / well in a 24-well plate. The seeding day was defined as "Day 0".

[0397] On day 2, replace the medium in the wells with the following:

[0398] Columns 1 and 2 were treated with in-house prepared spinal cord (SC) medium (the medium contained 0.6wt% D-glucose, 2mM L-glutamine, gentamicin 25μg / ml, B27, N2, BSA 0.1mg / ml, Hepes, 10% FBS, DMEM / F12 and IGF-I, 50ng / ml) + 1mM calcium from ACC (total calcium concentration of 2mM); columns 3 and 4 were treated with SC medium + 1mM calcium from CaCl2; columns 5 and 6 were treated with SC medium. Both types of cells (MBA13 and MDX) were cultured for up to 10 days.

[0399] Several wells of each type were fixed (with 4% paraformaldehyde for 20 minutes) on days 5, 7 and 10 after medium exchange, ie, on days 7, 9 and 12 of the study.

[0400] Once the cells were fixed, two staining reagents were used to stain for extracellular calcium or bone deposition to assess osteoblast function: (i) Alizarin red (Sigma A55333) and (ii) alkaline phosphatase (DAKO BCIP / NBT substrate system K0598) as follows:

[0401] Alizarin Red Staining Procedure - pH adjusted to 4.2. Wash cells twice with PBS and fix with cold methanol for 5 minutes and then wash again with PBS. Use alizarin solution at a concentration of 2% for 15-20 minutes. After staining incubation, wash samples 2-3 times with water to remove nonspecific staining.

[0402] Alkaline phosphatase staining - Wash the cells 1 or 2 times with PBS and then fix them with 4% paraformaldehyde for 20 minutes. Then, wash them 3 times with PBS and after removing the PBS residue, cover the cells with 3 drops of BCIP / NBT kit solution, including an empty well without any cells as a control for quantitative determination. Alkaline phosphatase kit incubation was performed for 1 hour and then rinsed with distilled water.

[0403] result

[0404] Results are presented for cells cultured for 10 days and stained with both alizarin and alkaline phosphatase. Observations made 10 days ago detected hardly any calcium deposition in either staining method.

[0405] The condition of the cells was observed under an optical microscope (without fixation) at two time points, day 2 and day 4. In both observations, the condition of the cells seeded on the wells pre-coated with MSC attachment solution was poor; the cells were rounded. In contrast, the condition of the cells seeded on the wells pre-coated with MSC attachment solution was poor; the cells were rounded. Pre-coated with gelatin Hole The cells on the Types of Pre-coating The following results and staining procedures are for cells grown in gelatin used as an attachment matrix.

[0406] In Alizarin red staining, calcium deposits are detected based on the orange-red color.

[0407] Alizarin red staining showed very strong signals in osteoblast samples supplemented with ACC compared to those samples supplemented with CaCl2 which showed only weak signals ( Figures 9A-9C ). In addition to the signal, it can be seen in the figure that large plaques of calcium deposits are stained, which are not observed in any of the control treatments.

[0408] Alizarin red staining of cells grown in MSCgo fast medium also showed some calcium deposition staining, but the size and number of deposits were significantly lower and similar to the morphology and number observed for cells supplemented with CaCl2. No calcium deposition was observed for cells grown in MSC NutriStem+ supplement (MSC sup; data not shown). MSCNutriStem+sup culture medium is generally used to induce cell proliferation rather than differentiation. In fact, the number of cells observed was large, but no calcium deposition was observed.

[0409] This observation suggests that ACC treatment promotes the differentiation of MBA13 cells into osteoblasts and enhances cellular calcium deposition, that is, promotes their function.

[0410] Another independent marker of osteoblast differentiation is alkaline phosphatase. This enzyme is maximally expressed when the cells undergo matrix maturation. Alkaline phosphatase staining is used as a complementary method to monitor osteoblast differentiation and function to validate the results obtained by alizarin staining.

[0411] Alkaline phosphatase staining is shown in black, and the results are presented in Figures 10A-10CMBA13 cells treated with ACC showed a strong signal compared to the other treatments. In fact, alkaline phosphatase staining supported the better osteoblast differentiation in cultures treated with ACC.

[0412] Alizarin staining of MDX cells treated with ACC-enriched medium 10 days after seeding showed no major differences in staining between treatments (see Figures 11A-11C These results support that the Alizarin Red staining of osteoblasts is due to calcium deposition by osteoblasts and not to ACC deposition caused by the treatment itself (ie, it is not an experimental artifact).

[0413] Alkaline phosphatase staining of MDX cell line ( Figures 11D-11E ) demonstrated that no bone formation occurred. Interestingly, in alkaline phosphatase staining, enhanced myotube formation was observed in ACC-treated cells compared to cells grown in medium supplemented with CaCl2 or controls.

[0414] in conclusion

[0415] Two independent stainings, Alizarin Red and Alkaline Phosphatase, after 10 days of seeding in different media showed significantly stronger staining of osteoblast deposits grown in media supplemented with ACC compared to the controls used.

[0416] Large plaques of calcium deposits were observed when cells were grown in the presence of ACC, no such deposits were observed for cells grown in medium supplemented with CaCl2. Stronger signals may indicate greater function, as staining showed more plaque deposition. The results obtained from staining of MDX cells suggest that the plaques are not derived from the addition of ACC, but are a direct result of osteoblast function.

[0417] Example 20 - ACC for promoting sperm motility

[0418] Materials and methods

[0419] ACC suspension preparation

[0420] 36 ml of 3% calcium chloride solution was mixed with 4 ml of 0.3% citric acid solution and 10 ml of 0.5% triphosphate solution. Then 40 ml of 2% sodium carbonate solution was added to precipitate ACC. 10 ml of stabilizing solution containing 0.5% triphosphate was added to the ACC suspension to produce a stable ACC suspension. The elemental calcium concentration of the obtained suspension was 75 mM.

[0421] Sperm collection

[0422] Sperm was collected 3 times from the same ram (experiment 1) or 2 times from 3 rams (experiments 2 & 3) and assessed for concentration and motility at room temperature. Sperm was diluted to a concentration of 50 million sperm / ml in synthetic oviductal fluid (SOF) consisting of: 107.70 mM NaCl, 7.16 mM KCl, 1.19 mM KH2PO4, 1.71 mM CaCl2, 0.49 mM MgCl2, 25.07 mM NaHCO3, 3.30 mM sodium lactate and 1.50 mM glucose in Milli-Q water (10). Osmolarity was 270 mOsmol and pH was 7.55.

[0423] Sperm motility was then assessed using CASA (The riogenology, 2014, Vol. 81, No. 1, pp. 5-17) and cooled to 4°C at 1°C / min.

[0424] Upstream procedures

[0425] The upstream procedure was performed at 38°C, and the original sperm (10 ul) was placed in an eppendorf 1 ml vial and incubated in SOF medium inserted into a 0.25 ml pipette (CBS, France) for about 1 hour. After 40-60 minutes, the sperm entering the pipette was placed on a slide and evaluated by CASA.

[0426] result

[0427] The motility of freshly collected spermatozoa was tested as described in the Materials and Methods section with or without the addition of amorphous calcium carbonate. The results are presented in Table 17.

[0428] Table 17. Evaluation of sperm motility with and without the addition of ACC suspension 34 μl / ml (2.6 mM elemental Ca).

[0429] % Motility % forward motility Comparison 96±1.15 55.3±1.15 ACC (34 μl / ml: 2.6 mM elemental Ca) 95±1 70.6±3.2

[0430] It can be seen that sperm supplemented with ACC have much higher progressive motility than untreated sperm.

[0431] Example 21. Effect of ACC on sperm motility in the swim up experiment

[0432] To evaluate the effect of ACC on sperm motility, a standard swim-up experiment was performed in the presence of 17 μl / ml and 34 μl / ml ACC suspensions (1.3 mM and 2.6 mM elemental Ca, respectively). Motile sperm were counted after 1 hour and the results are summarized in Table 18.

[0433] Table 18: Effect of ACC on sperm motility in the swim-up assay (concentration in millions / ml)

[0434]

[0435] It can be clearly seen that the addition of ACC significantly increased the number of viable sperm in all experiments. In samples supplemented with ACC, the number of viable sperm was 2 to 7 times higher than in untreated samples.

[0436] Example 22. Effect of ACC concentration on sperm motility in the upstream assay

[0437] The effect of ACC concentration on sperm motility was tested by a swim-up assay using 3 different samples for each ACC concentration. The results are summarized in Table 19.

[0438] Table 19. Effect of ACC concentration on sperm motility in the swim-up assay (concentration in millions / ml)

[0439]

[0440] It has been noted that the addition of 17 μl / ml ACC suspension in synthetic oviductal fluid (SOF) solution and incubation at 38°C for 1 hour increased the concentration of motile sperm by at least 3 times. Interestingly, higher concentrations of ACC did not reduce the concentration of motile sperm after swim-up. In conclusion, ACC increases sperm concentration after swim-up procedure and does not have any biphasic effect on ionized calcium. Any ACC concentration above the minimum effective dose enhances motility.

[0441] Example 23. ACC preserves ovaries in vitro

[0442] Materials and methods

[0443] Ovaries were collected from mice (6 weeks old) and cut into 0.4 mm × 0.4 mm fragments. The ovaries were cultured in 12-well plates. The culture medium included: 90% Dulbecco's modified eagle medium-nutrient mixture F-12 (DMEM-F12) decalcified (medium without calcium ions, specific preparation), 10% fetal bovine serum (FBS), 2mM glutamine, 25μg / mL gentamicin and 0.3-0.5% NVR-Gel, with or without stable 3.4mMACC-PP. After 48h of culture, 5IU of pregnant mare's serum gonadotropin (PMSG) was added to the culture dish.

[0444] result

[0445] from Figures 12A-12BIt can be seen that in the presence of stable ACC, after 2 weeks of in vitro culture, secondary follicles develop, and the granulosa cells around the oocytes are complete. In contrast, the secondary follicles of the control group show incomplete granulosa cells and germinal vesicles oocytes. In the ACC group, many more follicles are observed, meaning that adding ACC in the culture medium allows faster and better growth of follicles, as well as improved ovarian preservation.

[0446] Although the present invention has been described above by way of preferred embodiments thereof, modifications may be made thereto without departing from the spirit and nature of the invention as defined in the appended claims.

Claims

1. A cell culture medium, wherein the cell culture medium is supplemented with amorphous calcium carbonate (ACC) stabilized by at least one stabilizer, and wherein the cell culture medium is suitable for the growth of and promotes the growth of mammalian cell cultures, and wherein the stabilizer is selected from the group consisting of: inorganic polyphosphates containing 2 to 10 phosphate groups, phosphorylated amino acids, organic acids, phosphorylated, phosphonated, sulfated or sulfonated organic compounds, phosphates or sulfates of hydroxycarboxylic acids, bisphosphonates, and any combination thereof, wherein the mammalian cell culture is selected from the group consisting of cell cultures, tissue cultures, organ cultures, stem cells and organs of human or non-human mammals, and wherein the mammalian cell culture is selected from the group consisting of neural, muscle, epithelial, bone, fat, stem cell and blood cell cultures.

2. The cell culture medium of claim 1, wherein the growth of the biological culture is selected from at least one of proliferation, maturation, propagation, regeneration, cryopreservation or differentiation of the biological culture.

3. The cell culture medium according to any one of claims 1 to 2, wherein the cell culture is a primary cell culture or a cell line selected from a finite cell line and a continuous cell line.

4. The cell culture medium according to claim 3, wherein the cell line is selected from the group consisting of FM3, HeLa, 293, A-549, ALC, CHO, HB54, HL60, COS-7, HEK293, VERO, BHK, CV1, MDCK, 3T3, C127, MRC-5, BAE-1, SH-SY5Y, L-929, HEP G2, NSO, U937, NAMALWA, WEHI 231, YAC 1 and U 266B1 cell lines.

5. The cell culture medium according to claim 1, wherein the mammalian tissue culture is selected from the group consisting of epithelial tissue culture, connective tissue culture, muscle tissue culture and neural tissue culture.

6. The cell culture medium of claim 5, wherein the epithelial tissue culture is selected from the group consisting of skin, stomach and intestinal lining, kidney and glandular tissue cultures, the muscle tissue culture is selected from the group consisting of smooth muscle, skeletal muscle and cardiac muscle tissue cultures, and the neural tissue culture is selected from the group consisting of brain, spinal cord and neural tissue.

7. The cell culture medium according to claim 1, wherein the stem cells are selected from embryonic stem cells, chorionic stem cells, amniotic membrane stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, NOM stem cells, glial stem cells, adult stem cells and induced pluripotent stem cells.

8. The cell culture medium according to claim 1, wherein the culture medium is a natural culture medium selected from the group consisting of biological fluids, tissue extracts and clots; or an artificial culture medium selected from the group consisting of balanced salt solutions, basal medium and complex medium.

9. The cell culture medium according to claim 8, wherein the culture medium is a serum-free culture medium.

10. The cell culture medium according to claim 8 or 9, wherein the cell culture medium is selected from DMEM / F-12, cleavage medium, DMEM, RPMI 1640, MEM, IMDM, L-15 medium (Leibovitz), MCDB medium, medium 199, opti-MEM, Schneider Drosophila medium, Grace insect medium, IPL-41 insect medium Sf-900, serum-free insect medium, Shields and Sang M3 insect medium, TC-100 insect medium, TNM-FH insect medium, Ham's F-12, Ham's F-10, GMEM, Ames medium, Eagle's basic medium (BME), Claycomb, Click medium, Glasgow's minimum essential medium (GMEM), MegaCell medium, McCoy's 5A modified medium, NCTC medium, Williams medium E, Waymouth medium or TC-10 IPL-10 medium.

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