J Korean Soc Spine Surg.  2010 Jun;17(2):57-65. 10.4184/jkss.2010.17.2.57.

Adequate Serial Monolayer Passage Number of Human Intervertebral Disc Cells for Cell Therapy: Growth and Phenotype of Cells

• Kim YC
• Kim KB
• Park MS
• Kim SW

• KMID: 1459269
• DOI: http://doi.org/10.4184/jkss.2010.17.2.57

Abstract

STUDY DESIGN: This is an in-vitro experimental study.
OBJECTIVES
We wanted to analyze the changes in the growth and phenotype of human degenerative intervertebral disc cells depending on the frequency of subculture in an in vitro monolayer culture system. SUMMARY OF THE LITERATURE REVIEW: A subculture of disc cells is needed to obtain an adequate amount of disc cells for cell therapy, tissue engineering and analysis of the biological characteristics of degenerative disc cells.
MATERIALS AND METHODS
The obtained intervertebral discs were divided into the nucleus pulposus (NP) and the annulus fibrosus (AF). The AF and NP cells were cultured in a monolayer manner, respectively. At each subculture time, we analyzed the morphological changes, the adhesion rate, the proliferation rate and the viability. The expressions of types I and II collagen and proteoglycan were analyzed at the mRNA gene level.
RESULTS
Both the AF and NP cells gradually showed a fibroblast-like spindle shape while undergoing subculture. The adhesion rate was higher at the second and third times of subculture. The cell proliferation was the highest at the second subculture time. The viability was markedly lower prior to the subculture. On RT-PCR, the type II collagen expression was gradually decreased in the NP cells. In the AF cells, Type II collagen was not expressed from the second time of subculture. The expression of proteoglycan was gradually decreased in both.
CONCLUSIONS
Following the 3rd subculture, the degenerative disc cells had completely changed their original growth and phenotypic characteristics. Therefore, we believe that it is not desirable for us to do passage cultures more than three times for cell therapy.

Keyword

Intervertebral disc; Degenerative change; Phenotype; Subculture

MeSH Terms

Cell Proliferation
Collagen
Collagen Type II
Humans
Intervertebral Disc
Intervertebral Disc Degeneration
Phenotype
Population Characteristics
Proteoglycans
RNA, Messenger
Tissue Engineering
Tissue Therapy
Collagen
Collagen Type II
Proteoglycans
RNA, Messenger

Figure

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  Fig. 1Digital image of the obtained AF and NP tissue from degenerated intervertebral disc.

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  Fig. 2. The morphology of the AF cells and NP cells grown in the monolayer culture system. (x100)

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  Fig. 3. The adhesion ratio of the AF cells and NP cells at different passage number. (A) P0, (B) P1, (C) P2, (D) P3.

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  Fig. 4. Proliferation (%) of the AF cells and NP cells at different passage number. (A) AF cells, (B) NP cells.

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  Fig. 5. Viability of the AF cells and NP cells at different passage number.

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  Fig. 6. The mRNA expression of AF cells and NP cells at different passage number.

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  Fig 7. Measurement of the total collagen (A) and proteoglycan (B) in the culture medium of different passage number.

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