Method of co-culturing mammalian muscle cells and motoneurons
View Patent ↗The invention provides a method of co-culturing mammalian muscle cells and mammalian motoneurons. The method comprises preparing one or more carriers coated with a covalently bonded monolayer of trimethoxysilylpropyl diethylenetriamine (DETA); suspending isolated fetal mammalian skeletal muscle cells in serum-free medium according to medium composition 1; suspending isolated fetal mammalian spinal motoneurons in serum-free medium according to medium composition 1; plating the suspended muscle cells onto the one or more carriers at a predetermined density and allowing the muscle cells to attach; plating the suspended motoneurons at a predetermined density onto the one or more carriers and allowing the motoneurons to attach; covering the one or more carriers with a mixture of medium composition 1 and medium composition 2; and incubating the carriers covered in the media mixture.
1. A method of co-culturing muscle cells and motoneurons, the method comprising:
plating muscle cells and motoneurons onto a carrier surface,
covering the muscle cells and motoneurons in a first serum free medium, and
replacing the first serum free medium with a second serum free medium, the second serum free medium comprising a mixture of Neurobasal medium, B27, and GlutaMax, wherein the second serum free medium further comprises creatine, estrogen, and cholesterol.
2. The method of claim 1 , wherein replacing the first serum free medium with the second serum free medium occurs within one week of plating the muscle cells and motoneurons onto a carrier surface.
3. The method of claim 2 , further comprising periodically replenishing the second serum free medium for a maintenance period greater than 10 days.
4. The method of claim 1 , wherein the second serum free medium is devoid of growth factors.
5. The method of claim 1 , further comprising pretreating the carrier surface by depositing a non-biological growth substrate onto the carrier surface prior to plating the muscle cells and the motoneurons.
6. The method of claim 5 , wherein the non-biological growth substrate is a silicon based monolayer.
7. The method of claim 6 , wherein the non-biological growth substrate is trimethoxysilylpropyl diethylenetriamine (DETA).
8. The method of claim 1 , wherein the muscle cells are rat muscle cells.
9. The method of claim 1 , wherein the motoneurons are rat motoneurons.
10. The method of claim 1 , wherein the muscle cells are plated at a density of from about 700 cells/mm 2 to about 1000 cells/mm 2 .
11. The method of claim 1 , wherein the motoneurons are plated at a density of about 100 cells/mm 2 .
12. The method of claim 1 , wherein plating muscle cells and motoneurons comprises plating the muscle cells in a first step and later plating the motoneurons in a second step.
13. A method of co-culturing muscle cells and motoneurons, the method comprising:
plating muscle cells and motoneurons onto a carrier surface,
covering the muscle cells and motoneurons in a first serum free medium, and
replacing the first serum free medium with a second serum free medium,
wherein the second serum free medium comprises a mixture of Neurobasal medium, B27, and GlutaMax, and
wherein the first serum free medium is a mixture of Neurobasal Medium, antibiotic-antimycotic, Glutamax, B27 supplement, G-5 Supplement, vascular endothelial growth factor (VEGF) 165 rHuman , acidic FGF, heparin sulphate, leukemia inhibitor factor (LIF), vitronectin, ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT3), neurotrophin-4 (NT4), glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), cardiotrophin-1 (CT-1), cholesterol, human tumor necrosis factor alpha (TNF-alpha), platelet-derived growth factor-BB (PDGF-BB), vasoactive intestinal peptide (VIP), insulin-like growth factor-1, NAP, recombinant apolipoprotein E2, laminin, beta amyloid, human tenascin-C protein, sonic hedgehog (Shh) N-terminal protein, and agrin C-terminal protein.
14. The method of claim 13 , wherein the first serum free medium is a mixture of the composition of Medium 1 and the composition of Medium 2, wherein the composition of medium 1 is:
No.
Component(s)
Amount
1
Neurobasal Medium
500
mL
2
Antibiotic-Antimycotic
5
mL
3
Glutamax
5
mL
4
B27 Supplement
10
mL
5
G-5 Supplement (100×)
5
mL
6
VEGF 165 rHuman
10
μg
7
Acidic FGF
12.5
μg
8
Heparin Sulphate
50
μg
9
LIF
10
μg
10
Vitronectin (Rat Plasma)
50
μg
11
CNTF
20
μg
12
NT3
10
μg
13
NT4
10
μg
14
GDNF
10
μg
15
BDNF
10
μg
16
CT-1
10
μg
And wherein the composition of Medium 2 is:
No.
Component(s)
Amount
1
Neurobasal Medium
500
mL
2
Glutamax
5
mL
3
Antibiotic-Antimycotic
5
mL
4
B27 Supplement
10
mL
5
Cholesterol (250×)
5
mL
6
TNF-alpha, human
10
μg
7
PDGF BB
50
μg
8
VIP
250
μg
9
Insulin-like growth factor-1
25
μg
10
NAP
1
mg
11
Recombinant apolipoprotein E2
50
μg
12
Laminin, mouse purified
2
mg
13
Beta amyloid (1-40)
1
mg
14
Human Tenascin-C protein
100
μg
15
rr-Sonic hedgehog (Shh), N-
50
μg
terminal protein
16
rr-Agrin, C-terminal protein
50
μg.
15. The method of claim 13 , wherein the second serum free medium further comprises creatine, estrogen, and cholesterol.
16. The method of claim 13 , wherein replacing the first serum free medium with the second serum free medium occurs within one week of plating the muscle cells and motoneurons onto a carrier surface.
17. The method of claim 16 , further comprising periodically replenishing the second serum free medium for a maintenance period greater than 10 days.
18. The method of claim 13 , wherein the second serum free medium is devoid of growth factors.
19. The method of claim 13 , further comprising pretreating the carrier surface by depositing a non-biological growth substrate onto the carrier surface prior to plating the muscle cells and the motoneurons.
20. The method of claim 19 , wherein the non-biological growth substrate is a silicon based monolayer.
21. The method of claim 20 , wherein the non-biological growth substrate is trimethoxysilylpropyl diethylenetriamine (DETA).
22. The method of claim 13 , wherein the muscle cells are rat muscle cells.
23. The method of claim 13 , wherein the motoneurons are rat motoneurons.
24. The method of claim 13 , wherein the muscle cells are plated at a density of from about 700 cells/mm 2 to about 1000 cells/mm 2 .
25. The method of claim 13 , wherein the motoneurons are plated at a density of about 100 cells/mm 2 .
26. The method of claim 13 , wherein plating muscle cells and motoneurons comprises plating the muscle cells in a first step and later plating the motoneurons in a second step.