Micromass culture of human anulus cells: morphology and extracellular matrix production.
METHODS
Micromass culture was assessed as a cell culture microenvironment for anulus cells from the human intervertebral disc.


OBJECTIVE
To determine whether the micromass culture technique might be useful for the culture of human anulus cells.


BACKGROUND
Culture of cells in micromass has been traditionally used as a method to culture chondrocytes in a three-dimensional (3D) microenvironment with specialized chondrocyte media which allows expression of the chondrocytic phenotype. Recently it has also been used for disc cell 3D culture.


METHODS
Following approval of our human subjects Institutional Review Board, cells isolated from human anulus intervertebral disc tissue was cultured in micromass culture under control conditions or with addition of 5 ng/mL transforming growth factor-beta (TGF-beta). Cultures were grown for 7 days, and then analyzed for morphology with light microscopy, for extracellular matrix (ECM) production with transmission electron microscopy and quantitative measurement of total sulfated proteoglycan production. Immunohistochemistry was also performed to assess types I and II collagen, decorin, keratan sulfate, and chondroitin sulfate content of ECM.


RESULTS
Human anulus cells form multilayered colonies when cultured with minimal media and 20% fetal bovine serum in the micromass methodology. Stimulation of ECM production occurs when 5 ng/mL TGF-beta was added to the micromass media. TGF-beta also significantly increased the production of sulfated proteoglycans (P = 0.026). Under both control and TGF-beta-supplementation, the resulting micromass formed by anulus cells is not as compact as the micromass which results when stem cells cultured in chondrogenic media. Ultrastructural studies showed the presence of apoptotic cells and the presence of peroxisomes within cells. Immunohistochemical studies on production of type I collagen, decorin and keratan sulfate showed that there was localized production of these ECM components in focal regions; chondroitin sulfate and type II collagen, however, showed a more uniform overall production by cells within the micromass.


CONCLUSIONS
Human anulus cells were successfully cultured under micromass conditions in nonchondrogenic media and with TGF-beta supplementation which increased ECM production. The resulting anulus cell micromass, however, was not as rounded or compact as that which occurs with routine chondrocyte micromass or stem cells induced into chondrocyte differentiation. The presence of peroxisomes noted on ultrastructural studies may reflect cell stress or uneven distribution of nutrition within the micromass during the 7-day micromass culture period. Immunohistochemical studies showed nonuniform ECM gene expression and production within the micromass, suggesting variable gene expression patterns with this culture method.
Department of Orthopaedic Surgery, Orthopaedic Research Biology, Carolinas Medical Center, Charlotte, NC 32861, USA. helen.gruber@carolinashealthcare.org
Helen E
HE
Gruber
Yin
Y
Chow
Gretchen L
GL
Hoelscher
Jane A
JA
Ingram
Natalia
N
Zinchenko
H James
HJ
Norton
Yubo
Y
Sun
Edward N
EN
Hanley
D000328:Adult; D000368:Aged; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005260:Female; D051445:Fibrocartilage / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007403:Intervertebral Disc / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D007405:Intervertebral Disc Displacement / Q000601:surgery*; D008297:Male; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D020675:Peroxisomes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D011509:Proteoglycans / Q000032:analysis / Q000378:metabolism; D016378:Tissue Transplantation / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D003470:Culture Media; D016326:Extracellular Matrix Proteins; D011509:Proteoglycans; D016212:Transforming Growth Factor beta