New use of a three-dimensional pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering.
METHODS
Human intervertebral disc cells were cultured in a new three-dimensional "pellet culture" system as an alternative to conventional alginate bead microspheres. Histologic, biochemical, and immunohistologic assays were performed to characterize this new culturing method for disc cells. The feasibility of using the pellet culture system to study effects of gene therapy was also assessed.


OBJECTIVE
To characterize a new and simpler, three-dimensional culture system for human intervertebral disc cells and to assess the feasibility of its use for gene therapy and tissue engineering studies.


BACKGROUND
The alginate microsphere three-dimensional culture system has been the most utilized culture method for disc cells, but it is technically difficult and has some disadvantages. Recently, the "pellet culture" method, a simpler three-dimensional culture system, was described for bone marrow stromal cells. This simpler method might be useful for in vitro and in vivo studies of disc cells as well as for delivery of exogenous genes.


METHODS
Isolated human intervertebral disc cells were centrifuged at low speeds to form aggregates and allowed to grow as pellets for up to 3 weeks. At various times these pellet cultures were analyzed grossly, histologically, and immunohistologically. Their ability to incorporate [35S]sulfate in their response to TGF-beta1 was also analyzed. The ability of these pellets to deliver and express exogenous genes in vivo was analyzed by implantation of pellet cultures in muscles of SCID mice.


RESULTS
Within several days the intervertebral disc cells were able to form mature three-dimensional aggregates that were each well encapsulated by a fibrous capsule. These pellets successfully synthesized proteoglycan and collagen Type II matrix as determined by histology and immunohistochemistry. In response to TGF-beta1, the pellets increased synthesis of proteoglycan and collagen Type II. When implanted into thigh muscles of SCID mice, the pellets remained aggregated and expressed the beta-galactosidase marker gene in vivo for up to 2 weeks.


CONCLUSIONS
The pellet culture system is a new and technically simple method to culture human intervertebral disc cells. The majority of the human intervertebral disc cells retained their native phenotype in this three dimensional system and expressed a marker gene both in vitro and in vivo. Thus, this pellet system might be useful for in vitro and in vivo biochemical studies as well as for studies involving gene therapy and tissue engineering.
Lee
J Y
JY
Department of Orthopaedic Surgery, CHildren's Hospital, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Hall
R
R
Pelinkovic
D
D
Cassinelli
E
E
Usas
A
A
Gilbertson
L
L
Huard
J
J
Kang
J
J
D000328:Adult; D000368:Aged; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D024043:Collagen Type II / Q000032:analysis / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000096:biosynthesis; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism; D005240:Feasibility Studies; D005260:Female; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism; D007405:Intervertebral Disc Displacement / Q000473:pathology / Q000601:surgery; D007763:Lac Operon / Q000235:genetics; D008297:Male; D051379:Mice; D016513:Mice, SCID; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000201:enzymology; D011509:Proteoglycans / Q000096:biosynthesis / Q000235:genetics; D013462:Sulfur Radioisotopes; D023822:Tissue Engineering / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D024043:Collagen Type II; D011509:Proteoglycans; D013462:Sulfur Radioisotopes; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D004247:DNA; D001616:beta-Galactosidase