WO2003012076A2

WO2003012076A2 - Culture medium to improve the purity of myoblast cultures and method using same

Info

Publication number: WO2003012076A2
Application number: PCT/CA2002/001216
Authority: WO
Inventors: Jacques P. Tremblay
Original assignee: Universite Laval
Priority date: 2001-08-02
Filing date: 2002-08-02
Publication date: 2003-02-13
Also published as: WO2003012076A3; CA2354978A1; AU2002322245A1

Abstract

The present invention relates to a novel culture medium composition to improve the percentage of myoblasts in a primary culture made from a muscle biopsy, which in turn permits a more successful treatment of myopathies and cardiopathies. The present invention also relates to a method to increase the preparation of myoblasts in a primary culture of mixed cells and in particular from a muscle biopsy. The invention also relates to such enriched myoblast cultures to treat myopathies and cardiopathies. In addition, the invention relates to a nutrient medium which increases the proportion of myoblasts with respect to other cell types. The present invention also relates to a myoblast basal growth medium comprising all nutrients required for growth and expansion of myoblasts wherein at least a portion of the amino acid L-valine is replaced with D-valine.

Description

TITLE OF THE INVENTION

CULTURE MEDIUM TO IMPROVE THE PURITY OF MYOBLAST CULTURES AND METHOD USING SAME

FIELD OF THE INVENTION

The present invention relates to a novel culture medium composition to improve the percentage of myoblasts in a primary culture made from a muscle biopsy, which in turn permits a more successful treatment of myopathies and cardiopathies. The present invention also relates to a method to increase the preparation of myoblasts in a primary culture of mixed cells and in particular from a muscle biopsy. The invention also relates to such enriched myoblast cultures to treat myopathies and cardiopathies. In addition, the invention relates to a nutrient medium which increases the proportion of myoblasts with respect to other cell types.

BACKGROUND OF THE INVENTION

Myoblast transplantation has been investigated for many years as a potential treatment of myopathies, particularly dystrophies. Myoblast transplantation is also a potential treatment for various cardiomyopathies and in particular heart insufficiency. Methods to proliferate myoblasts are known. For example, Ham et al. 5,324,656 teach a media for normal human muscle satellite cells termed MCDB120 which is a synthetic or semi-synthetic media. However, since Ham used clonally grown myoblasts (Karpati et al. 1990, Adv. Exp. Med. Biol. 208:31-34), a need to avoid the growth of fibroblasts or other cells was not addressed. Indeed, Ham et al. teach that 2because these studies were done with cloned human muscle satellite cells, they were focused exclusively on improvement of growth, with no immediate concern about expression of differentiated properties or overgrowth by fibroblasts", "moreover the present studies were done with clonal cultures and allowed optimization of the medium for growth without concern about differentiation or overgrowth by other cell types." It is also known that mitogens such as FGF and EGF have been reported to promote the growth of fibroblasts (Ham et al. supra;

Dollenmeir et al. 1981 , Exp. Cell Res. 135:47-61). Since a low percentage of fibroblasts is better to treat both myopathies and cardiomyopathies, because it avoids or lowers important fibrosis, there thus clearly is a need to identify a medium and methods using same which will favor myoblast growth and avoid in particular fibroblast growth, thereby enabling a better treatment upon transplantation of the myoblasts.

There thus remains a need to provide a culture medum which increases the proportion of myoblasts in culture relative to other cell types as well as to methods for enhancing the proportion of myoblasts relative to other cell types.

The stimulation of fibroblasts could pose an inconvenience to transplantation results. There remains a need to provide primary myoblast cultures which have low amounts of fibroblast and non- fibroblast proliferation inducing media could enable preconditioning of the donors' myoblasts for a longer time, thereby increasing the number of cells to be transplanted from a relatively small biopsy.

The present invention seeks to meet these and other needs. The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety. SUMMARY OF THE INVENTION

The present invention thus relates to a culture medium composition that favors the proliferation of myoblasts over that of other cell type such as in a non-clonally grown culture. In one particular embodiment, the culture media enhances the proliferation of myoblasts over that of the fibroblasts.

Transplantation or implantation is a promising therapy for a number of diseases. Transplantation can serve to replace cells that have been damaged by an intrinsic disease or otherwise. Transplantations can be autologous, i.e., the patient can serve as his or her own donor or not (e.g. histocompatible donor). To date, however, transplantation efficiencies have been hampered by the culturing methods and media available. The media and methods of the present invention seek to correct this defect.

As used herein, the terms "expansion" or "expanding" of cells mean a culturing of cells for a time and under conditions that allow the cells to grow and to multiply, thereby yielding a greater number of cells at the end of the expansion than at the beginning thereof. Herein, the term "passage" relates to the process whereby cells which have reached a certain number, or density, (e.g. confluence) are detached from the tissue culture support (e.g. flask), collected and resuspended in tissue culture medium. The suspension can be distributed to fresh tissue culture supports, (plates or flasks), so as to provide a greater total surface area for growth of the cells.

Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. Generally, the procedures for cell cultures, infection, molecular biology methods and the like are common methods used in the art. Such standard techniques can be found in reference manuals such as for example Sambrook et al. (1989, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratories) and Ausubel et al. (1994, Current Protocols in Molecular Biology, Wiley, New York).

It should be understood that for certain embodiments, the myoblasts could be genetically engineered so as to increase the expression of a chosen sequence or protein or alternatively to press the expression thereof. Methods to transfect cells and methods of genetically engineering a chosen cell or cell type are well-known in the art.

Broadly, the invention refers to a nutrient media which enables growth of myoblasts in culture in which a significant portion of L- valine has been replaced by D-valine, thereby favoring the growth of the myoblasts as compared to that of fibroblasts. In one particular embodiment, the medium is substantially devoid of L-valine and a macrophage-growth promoting amount of D-valine is added.

While Picciano et al. in Exp. Cell. Res. 151:134-147 (1984) reported that D-valine cannot be used by fibroblasts, nothing is disclosed in Picciano on the capacity of myoblasts to use D-valine. Indeed, Picciano used endothelial cells which were maintained in a medium comprising D-valine while contaminating fibroblasts were removed.

In one embodiment, the present invention provides a nutrient medium designated TREM-1 , which is optimized for the preferential growth of myoblasts over that of other cell types and more particularly fibroblasts. Tables 1 and 2 provide the composition of TREM- 1. It should be understood to a person of ordinary skill in the art of the present invention, that the composition of the TREM-1 medium can be adjusted to suit particular needs, as well as to further optimize improving capabilities of enhancing myoblast growth over that of other cell types and in particular fibroblasts. It should be understood that a variation of about 10% in the concentration of the different components of the TREM-1 medium (and of the supplements, such as found in Table 2) are considered to be variations which do not significantly affect the growth properties of the medium of the present invention.

It will be understood that the position of nutrients in TREM-1 is chosen so as to satisfy all cellular requirements for nutrients. The term "nutrient" refers to the defined chemical substance which can enter a cell and be used as a structural component, in energetic pathways, as a substrate in the biosynthesis of a particular molecule, group thereof, or as a co-factor for these processes. Non-limiting examples of nutrients include amino acids, vitamins, and carbohydrates. Other factors which are required for cellular proliferation are generally considered as "supplements". Supplements usually satisfy non-structural cellular growth requirement and often are necessary to enable growth of the cells in the basal nutrient medium. Non-limiting examples of supplements include growth factors, serum and cell or tissue extracts. Often, the additives are of an undefined nature, since they are obtained from serum or cell extracts. Thus, to a basal nutrient medium (which is considered "defined"), "undefined" factors are often added (e.g. serum or growth factors). Depending on the concentration of the added undefined supplement, the media could be considered a semi-defined media. While for certain applications it might be advantageous to use a defined medium, since all components are known, this is often impossible because undefined supplements are often required to enable growth of the cells in the defined basal medium. The term "semi-defined medium" as used herein refers to a medium which is capable of supporting the cell growth, but which contains a minimum amount of undefined components.

It will be understood that the optimization of the nutrient medium of the present invention can be carried-out by testing different concentrations of each of the given nutrients (or supplements). For example, myoblast cells can be grown in increment concentrations of a particular nutrient and qualitative and quantitative measurements of the growth of the cell can be carried-out. In one particular embodiment of the present invention, such quantitative and qualitative assessments of nutrient growth requirements of myoblasts are carried-out in parallel with requirements for fibroblasts and a quantitative and qualitative assessment of the growth of myoblasts over that of fibroblasts is carried-out. A non- limiting example of how nutrient requirements for growth can be assessed include assembling the TREM-1 medium without a particular nutrient, adding varying concentrations thereof and assessing myoblast growth (or myoblast versus fibroblast growth).

As known to a person of ordinary skill, growth of a cell with respect to the concentration of a given nutrient can be graphed. It is usually observed that the growth response follows a bell-shaped curve, which often includes a plateau region. This region is due to the fact that the requirement for the nutrient is no longer limiting for cell growth and the concentration is not high enough to be inhibitory. Of course, it is well- known that plateau region in the curve is dependent on the particular nutrient and of its requirement to a particular cell type, such that it can be broad or narrow. By using for example this type of curve, optimization of the nutrient concentrations can be adjusted. For example, using concentrations of a nutrient which are near or at the mid-point of their plateau (on a semi-logarithmic graph of optimum growth, for example). Example of such an optimization of the amino acid arginine for the growth media in human muscle satellite cells, MCDB120, is shown of Figure 1 of Ham et al. supra. It will be understood that certain nutrients can also be adjusted based on their known effective amounts in other mediums for other cell types, provided of course that they do not negatively affect the growth of myoblast cells and do not overcome the growth-limiting effects of the TREM-1 medium on fibroblasts.

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non- restrictive description of preferred embodiments which is exemplary and should not be interpreted as limiting the scope of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The culture medium (TREM-1) of the present invention was compared with a culture medium designed by Dr. Ham for myoblast proliferation (In Vitro Cellular and Developmental Biology. 24(8): 833-844, 1988) and described in 5,324,656. The similarities and differences in the compositions of these two culture media are summarized in Tables 1 and 2, respectively. The most striking difference between the two media is the replacement of L-Valine in the MCDB120 medium by D-Valine in TREM-1 to enrich myoblast cell populations. Fetuin was also eliminated in the TREM-1 medium. Another significant difference is that the Epidermal Growth Factor (EGF) present in the MCDB120 was replaced by the basic Fibroblast Growth Factor (bFGF) because it has been previously demonstrated that bFGF improves the success of myoblast transplantation (see US Patent Number 5,833,978). Table 1 : Common characteristics between the basal nutrients media MCDB120 and TREM-1

AMINO ACID mg/L

L-Alanine 2.67

L-Arginine.HCI 210.67

L-Asparagine.H2O 15.01

L-Aspartic Acid 13.31

L-Cysteine.HCI.H2O 35.13

L-Glutamic Acid 4.41

L-Glutamine 1461.50

Glycine 2.25

L-Histidine.HCI.H2O 41.93

L-lsoleucine 65.58

L-Leucine 131.17

L-Lysine.HCL 181.65

L-Methionine 29.84

L-Phenylalaline 33.04

L-Proline 11.51

L-Serine 31.53

L-Threonine 35.73

L-Tryptophan 4.08

L-Tyrosine 18.12

VITAMINS d-Biotin 0.00733

Folinic Acid (Ca salt).5H2O 0.602

DL-alpha-Lipoic Acid 0.002063

Niacinamide 6.11

D-Pantothenic Acid 23.82

(hemi-Ca salt)

Pyridoxine.HCL 2.056

Riboflavin 0.003764

Thiamin.HCL 3.373

Vitamin B12 0.01355 OTHER ORGANIC

COMPONENTS

Adenine 0.1351

Choline Chloride 13.96

D-Glucose 1000.00 myo-lnositol 18.016

Putrescine.2HCL 0.0001611

Sodium Pyruvate 110.04

Thymidine 0.02422

BULK INORGANIC SALTS

CaCI2.2H2O 235.23

KCI 298.20

MgSO4.7H2O 246.38

NaCI 6430.0

Na2HPO4.7H2O 134.04

TRACE ELEMENTS

CuSO4.5H2O 0.002496

FeSO4.7H2O 0.8340

H2SeO3 0.00387

MnSO4.5H2O 0.000241

Na2SiO3.9H2O 2.842

(NH4)6Mo7O24.4H2O 0.00371

NH4V03 0.000585

NiCI2.6H2O 0.0000713

ZnSO4.7H2O 0.08625

BUFFERS, INDICATORS

AND MISCELLENOUS

Phenol Red (Na salt) 1.242

NaHCO3 1176.0 Table 2: Differences between MCDB120 and TREM-1

In the basal nutrient medium

Composition of culture medium TREM-1 MCDB120 per mL per mL

D-Valine 0.117 mg

L-Valine 0.117 mg

In the supplements added to the basal medium

Bovine serum albumin 0.5 mg 0.5 mg

Dexamethasone 0.39 μg 0.39 μg

Insulin 5 μg 180 μg

Fetuin (traction of fetal bovine serum) 0 mg 0.5 mg

Basic Fibroblast Growth Factor (bFGF) 10 ng O ng

Epidermial Growth Factor (EGF) O ng 10 ng

Streptomycin 100 μg 100 μg

Penicillin 100 U 100 U

Fetal bovine serum (FBS) 0.15 mL 0.15 mL

The present invention is illustrated in further detail by the following non-limiting example.

EXAMPLE 1 To demonstrate the usefulness of the TREM-1 culture medium relative to the MCDB120 culture medium, myoblast cultures from four (4) healthy individuals were prepared. The number of cells after the primary culture and after the first and second passage of the culture was determined by counting cell samples from each individual with an hemacytometer. The percentage of myoblasts in the culture was determined at the same time by staining the cells with a mAb specific for NKH-1, a cell surface protein expressed by the myoblasts but not by the fibroblasts. At each passage, five hundred thousand (500,000) cells were transferred to a 75 cm³ culture flask in order to calculate the number of cells produced at each passage. The results are presented in Table 3. As can be seen, there is a significant increase in the percentage of myoblasts in the culture comprising TREM-1 medium as compared to the MCDB120 medium.

Table 3: Myoblast Production

TREM-1 MCDB-120

Individual Nb of % Nb of % number cells myoblasts cells myoblasts

X X

1 ,000,000 1 ,000,000

Individual 1 Primo 0,66 92 1 76 culture

Passage 1 11 ,22 89 24 76

Passage 2 181 ,76 97 408 87

Individual 2 Primo 0,66 6 culture

Passage 1 2,5 45 4,88 11

Passage 2 12 27 84,91 20

Individual 3 Primo 1 ,1 94 1 ,2 91 culture

Passage 1 19,4 99 17,28 93

Passage 2 426,8 98 387,07 96

Individual 4 Primo 2,2 71 2 50 culture

Passage 1 58,52 66 35,6 46

Passage 2 971 ,43 69 178 69 The formation of muscle fibers following the transplantation of myoblasts grown in each of the two media was compared by transplanting three (3) million cells from each culture into the Tibialis anterior muscle of immunodeficient SCID mice. The number of muscle fibers expressing human dystrophin was established a month after the transplantation by staining cryostat muscle cross-sections with the mAb NCIDys3 (Novocastra inc.) which reacts with human dystrophin but not with mouse dystrophin (results in Table 4). Significantly more muscle fibers expressing human dystrophin appeared following the transplantation of myoblasts grown in TREM-1 than in MCDB120.

Table 4: Muscle Fibers Expressing Human Dystrophin

Nb of dystrophin positive

Individual fibers number

TREM-1 MCDB-120

Individual 1 150 64

Individual 2 48 39

Individual 3 93 121

Individual 4 339 81

Thus, the present invention also relates to a method of increasing the transplantation frequency and/or of increasing the proportion of myoblasts which express a chosen factor (e.g. human dystrophin) upon transplantation; and/or increase the proportion of muscle-fibers expressing dystrophin upon transplantation, comprising a growing of myoblasts in a culture medium of the present invention which favors the growth of myoblasts over that of fibroblasts and transplanting the myoblasts in the patient. Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified without departing from the spirit and nature of the subject invention as defined in the appended claims.

REFERENCES

Ham et al. 1988, In Vitro Cellular and Developmental Biology 24(8): 833- 844.

Ham et al. Improved media for rapid clonal growth. Myoblast Transfer Therapy, pp 193-199, Edited by R. Griggs and G. Karpati. Plenum Press, New York 1990.

Picciano et al. 1984, Exp.. Cell Research 151 :134-147.

Claims

WHAT IS CLAIMED IS:

1. A myoblast basal growth medium comprising all nutrients required for growth and expansion of myoblasts and wherein at least a portion of the amino acid L-valine is replaced with D-valine.

2. The myoblast basal growth medium of claim 1 , wherein said medium is substantially devoid of L-valine.

3. The myoblast basal growth medium of claim 1 or 2, further comprising an undefined supplement.

4. The myoblast basal growth medium of claim 1 , 2 or 3, wherein said undefined supplement comprises a component selected from the group consisting of bovine serum albumine (BSA), dexamethasone, insulin, fetuin, basic fibroblast growth factor (bFGF), epidermial growth factor (EGF), fetal bovine serum (FBS) and any combination thereof.

5. The myoblast basal growth medium of claim 4, wherein said supplement is substantially devoid of fetuin and EGF.

6. The myoblast basal growth medium of claim 5, wherein said supplement contains a myoblast growth-promoting amount of bFGF.

7. The myoblast basal growth medium of claim 6, wherein said supplement contains a minimal amount of EGF.

8. A method for favoring the in vitro growth of myoblasts over that of fibroblasts comprising growing a mixture of cells containing myoblasts and fibroblasts in the myoblast basal growth medium of claim 1 , wherein the replacement of L-valine with D-valine favors the growth of myoblasts over that of fibroblasts.

9. The method of claim 8, wherein said mixture is obtained from a sample or biopsy of an individual.

10. A method for increasing a formation of muscle fibers following transplantation of myoblasts into a patient comprising in vitro culturing myoblasts from a patient according to the method of claim 8 or 9, and transplanting said myoblasts grown in vitro into said patient.