Optimizing nonviral-mediated transfection of human intervertebral disc chondrocytes.
BACKGROUND
The use of viral vectors for transfection of human disc chondrocytes has been well documented. However, because of immunological and cell toxicity concerns, nonviral reagents may provide gene delivery to intervertebral disc (IVD) chondrocytes without these associated obstacles. Several studies have been done using nonviral delivery systems with varying degrees of success.


OBJECTIVE
The purpose of the study was to determine the efficiency, toxicity, and optimal conditions for gene delivery into human degenerative IVD cells via nonviral reagents in vitro.


METHODS
In vitro viral and nonviral gene transfer.


METHODS
Human disc chondrocytes from 21 patients undergoing discectomy for trauma, disc herniation, and fusion for scoliosis or degenerative low back pain.


METHODS
Cell cytotoxicity and transfection efficiency as determined by microscopy, luciferase assay, and flow cytometry.


METHODS
Seventeen lipid-based nonviral reagents coupled to DNA plasmids coding for luciferase were transfected into cultured chondrocytes. Cells were transfected with varying ratios of DNA plasmid to reagent, harvested at 48 hours and analyzed for transfection rates and cell viability. Transfections with adenoviral constructs were comparisons. The three most efficient reagents were then coupled to green fluorescent protein and the experiments repeated. The most efficient reagent after these experiments (LT1) was tested in standard chondrocyte-maintenance medium and a minimal medium mixture devoid of antibiotics, buffers, and amino acids. Finally, LT1 in minimal medium with various hyaluronidase treatments was tested. The most effective reagents and relative toxicity as measured by flow cytometry were analyzed using repeated measures analysis of variance.


RESULTS
LT1 was most efficient and least toxic of nonviral reagents tested. LT1 had a mean percent survival of 78.1% versus 26.6% for TKO, 15.8% for T-Jurkat, and 70.8% in controls. Transfection was 1.5%. LT1 in minimal medium was significantly better than other reagents for both cell viability and transfection percentages. Minimal medium increased transfection with other reagents, yet cell viability with TKO and T-Jurkat was poor. Hyaluronidase had no effect on the viability of controls and decreased viability from 74.9% to an overall mean of 62.6% for all treatments. Transfection percentages increased from 1.8% without treatment to 15.2% with 40 units and 10.4% with four units of hyaluronidase given 24 hours before transfection and left in throughout the experiment. When treated at the time of transfection, efficiency was not significantly different to samples without hyaluronidase added. Additionally, hyaluronidase added 24 hours before transfection and washed out at the time of transfection significantly increased transfection percentages.


CONCLUSIONS
LT1 was the most efficient reagent in terms of transfection ability and cell toxicity compared with other reagents. Treatments in minimal medium yielded significant increases in transfection and no significant difference in toxicity as compared with controls. Hyaluronidase treatments improve transfection significantly but also increase toxicity. These results suggest that the nonviral reagent LT1 can be used to transfect IVD chondrocytes in vitro and may help facilitate gene transfection of IVD chondrocytes in vivo.
Morrey
M E
ME
Department of Orthopedics and Rehabilitation, University of Wisconsin Hospital and Clinics, School of Medicine and Public Health, Madison, WI 53792, USA. morrey.mark@mayo.edu
Anderson
P A
PA
Chambers
G
G
Paul
R
R
D019902:Chondrocytes / Q000502:physiology*; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006657:Histones / Q000627:therapeutic use; D006801:Humans; D007403:Intervertebral Disc / Q000502:physiology*; D008055:Lipids / Q000627:therapeutic use; D014162:Transfection / Q000379:methods*
D006657:Histones; D008055:Lipids