Muscle
LHCN-M2

In vitro propagation

Passaging of cells

The new culture flasks have to be pre-coated with porcine gelatin. Therefore, the culture flasks are treated with Gelatin solution (80 µl/cm²) at 37°C for at least 4 hours (up to one week).

Before introducing cells, remove excess of Gelatin solution. Use the pre-coated flasks immediately for seeding of cells, the surface must not dry out.For detachment of the cells remove and discard the culture medium and wash the cells once with PBS.Remove PBS completely.Then, add Trypsin-EDTA solution (20 µl/cm²), make sure that all cells have been in contact with Trypsin-EDTA and incubate the culture flask at 37°C for approximately 2 – 3 min.Observe cell detachment under an inverted microscope. As soon as all cells are detached, add growth medium (about 160 µl/cm²) and aspirate cells by pipetting.Determine the viable cell number and add appropriate aliquots of the cell suspension to new Gelatin coated culture vessels filled with growth medium (final volume of 240 µl/cm²).A seeding density of 1200 cells/cm² is recommended.Cells should be split twice a week when having reached about 30 – 40 % confluence. Never allow the culture to become confluent!Cultivate cells at 37°C in a humidified atmosphere with 5% CO2.

Cryopreservation

Freezing of cells

Detach the cells from the culture vessel by using Trypsin-EDTA solution (Protocol passaging of LHCN-M2).

Resuspend the detached cells in growth medium MyoUp and centrifuge at 170 g for 5 min.Then, discard the supernatant, resuspend the cell pellet in the remaining droplet.Add freezing medium (tempered to 4°C) to reach a cell density of 5000 – 6000 cells/cm² (3.75 – 4.5 x 10^5 cells for thawing in a 75 cm² culture flask).Add 1 ml of this cell suspension to each pre-cooled cryovial and immediately transfer the cells to -80°C.After 24 hours transfer the vials to the liquid nitrogen tank.

Thawing of cells

Original Evercyte cells are to be thawed in a T75 roux flask

Pre-coat a 75 cm² culture flask with gelatin (Protocol passaging of LHCN-M2).Add 15 ml of growth medium to the pre-coated 75 cm² culture flask and place the culture flask in the incubator for at least 30 min to allow the medium to reach its normal pH.Take a vial of frozen cells, rinse it outside with Ethanol and pre-warm in hand until one last piece of frozen cells is seen.Then, immediately transfer the content of the vial to a 15 ml centrifugation tube pre-filled with 9 ml of medium pre-cooled to 4°C and centrifuge for 5 min at 170 g.Discard the supernatant and resuspend the cell pellet in the remaining droplet.Add 8 ml of pre-warmed medium to the cell suspension, transfer the cells to the prepared culture flask and incubate at 37°C in a suitable incubator.Perform a medium change 24 hours after thawing. If the cells are already 30 – 40 % confluent at this point, they have to be passaged.

Product data sheet – certificate of analysis

is available upon request | Please contact us indicating the respective LOT numbers

Protocols

Data on Markers and Functions

LHCN-M2 – NGS data of undifferentiated cells generated by the Human Protein Atlas LHCN-M2 – part of ENCODE project, UCSF genome assembly

Safety documents (coming soon)

Selected publications

 

Tan A, Younis AZ, Evans A, Creighton JV, Coveny C, Boocock DJ, Sale C, Lavery GG, Coutts AS, Doig CL. PARP1 mediated PARylation contributes to myogenic progression and glucocorticoid transcriptional response. Cell Death Discov. 2023 Apr 22;9(1):133. doi: 10.1038/s41420-023-01420-2. 

Turner MC, Brett R, Saini A, Stewart CE, Renshaw D. Serum concentration impacts myosin heavy chain expression but not cellular respiration in human LHCN-M2 myoblasts undergoing differentiation. Exp Physiol. 2023 Feb;108(2):169-176. doi: 10.1113/EP090564. 

García-Benlloch S, Revert-Ros F, Blesa JR, Alis R. MOTS-c promotes muscle differentiation in vitro. Peptides. 2022 Sep;155:170840. doi: 10.1016/j.peptides.2022.170840. 

Gludovacz E, Schuetzenberger K, Resch M, Tillmann K, Petroczi K, Vondra S, Vakal S, Schosserer M, Virgolini N, Pollheimer J, Salminen TA, Jilma B, Borth N, Boehm T. Human diamine oxidase cellular binding and internalization in vitro and rapid clearance in vivo are not mediated by N-glycans but by heparan sulfate proteoglycan interactions. Glycobiology. 2021 May 3;31(4):444-458. doi: 10.1093/glycob/cwaa090. PMID: 32985651.

Maurer M, et al. (2015), IL-6 and Akt are involved in muscular pathogenesis in myasthenia gravis. Acta Neuropathol Commun. 2015 Jan 15;3:1. [PMID: 25627031] https://pubmed.ncbi.nlm.nih.gov/25627031/

Meyer S.U. et. al (2015), TNF-α and IGF1 modify the microRNA signature in skeletal muscle cell differentiation. Cell Commun Signal. 2015 Jan 29;13:4. [PMID: 25630602]https://pubmed.ncbi.nlm.nih.gov/25630602/

Salvadó, L., et. al. (2014), PPARβ/δ prevents endoplasmic reticulum stress-associated inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent mechanism. Diabetologia. 2014 Oct;57(10):2126-35. [PMID: 25063273] https://pubmed.ncbi.nlm.nih.gov/25063273/Salvadó, L., et al. (2013), Oleate prevents saturated-fatty-acid-induced ER stress, inflammation and insulin resistance in skeletal muscle cells through an AMPK-dependent
mechanism. Diabetologia. 2013 Jun;56(6):1372-82. [PMID: 23460021] https://pubmed.ncbi.nlm.nih.gov/23460021/Zhu, Ch. et al. (2008), SGNP: an essential Stress Granule/Nucleolar Protein potentially involved in 5.8s rRNA processing/transport. PLoS One. 2008;3(11):e3716. [PMID: 19005571] https://pubmed.ncbi.nlm.nih.gov/19005571/Roumes, H. et al. (2010), Calpains: markers of tumor aggressiveness?, Exp Cell Res. 2010 May 15;316(9):1587-99. [PMID: 20193680] https://pubmed.ncbi.nlm.nih.gov/20193680/Zhu, Ch.-H. et al. (2007), Cellular senescence in human myoblasts is overcome by human telomerase reverse transcriptase and cyclindependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies, Aging Cell, 6(4):515-23,[PMID: 17559502] https://pubmed.ncbi.nlm.nih.gov/17559502/

Licence Conditions

The business concept of Evercyte is to out-license telomerized cells to our customers. The license conditions depend on whether the contract partner is a for profit or a nonprofit organization and the intended use of the cells.

Nonprofit organizations

Evercyte grants licenses for an unlimited period to academic or nonprofit-organizations, whereby the use of Evercyte cell lines is restricted to research & development purposes and non-commercial use. The cells are not intended for human use. The customers have to agree to the conditions described in our material transfer agreement as well as accept our general terms and conditions.

On time payment for unlimited use: EUR 1600

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Profit organizations

Pharmaceutical – chemical- cosmetic industries

Evercyte grants licenses for commercial organizations, whereby we offer an initial testing phase for a flat fee that allows our customers to test our cells in their laboratories for a period of 6 months. Thereafter, annual license fees fall due, depending on the cell line of interest. Besides offering cell lines for research & development purposes, we also have established cell factories that qualify for production of clinical grade extracellular vesicles for human application. The customer has to agree to the conditions described in our license agreements.

Contract research organizations (CRO)

Evercyte grants licenses for contract research organizations, whereby we offer an initial testing phase for a flat fee that allows our customers to test our cells in their laboratories. Thereafter, we would negotiate a royalty based long-term license agreement individually. The use of the cells during these phases is restricted to research & development purposes. The cells are not intended for human use. The customers have to agree to the conditions described in our material transfer agreement and accept our general terms and conditions.

Initial license fee for 3 months: EUR 2500

Annual license fee R&D: royalty based