|Year : 1998 | Volume
| Issue : 1 | Page : 37-40
Effects of viscoelastic ophthalmic solutions on cell cultures
HN Madhavan, S Roy
Vision Research Foundation, Sankara Nethralaya, Chennai, India
H N Madhavan
Vision Research Foundation, Sankara Nethralaya, Chennai
Source of Support: None, Conflict of Interest: None
The development of mild but significant inflammation probably attributable to viscoelastic ophthalmic solutions in cataract surgery was recently brought to the notice of the authors, and hence a study of the effects of these solutions available in India, on cell cultures was undertaken. We studied the effects of 6 viscoelastic ophthalmic solutions (2 sodium hyaluronate designated as A and B, and 4 hydroxypropylmethylcellulose designated as C, D, E and F) on HeLa, Vero and BHK-21 cell lines in tissue culture microtitre plates using undiluted, 1:10 and 1:100 dilutions of the solutions, and in cover slip cultures using undiluted solutions. Phase contrast microscopic examination of the solutions was also done to determine the presence of floating particles. The products D and F produced cytotoxic changes in HeLa cell line and these products also showed the presence of floating particles under phase contrast microscopy. Other products did not have any adverse effects on the cell lines nor did they show floating particles. The viscoelastic ophthalmic pharmaceutical products designated D and F have cytotoxic effects on HeLa cell line which appears to be a useful cell line for testing these products for their toxicity. The presence of particulate materials in products D and F indicates that the methods used for purification of the solution are not effective.
Keywords: Viscoelastic ophthalmic solution, cell culture, cytotoxicity, sodium hyaluronate, hydroxypropylmethylcellulose
|How to cite this article:|
Madhavan H N, Roy S. Effects of viscoelastic ophthalmic solutions on cell cultures. Indian J Ophthalmol 1998;46:37-40
|How to cite this URL:|
Madhavan H N, Roy S. Effects of viscoelastic ophthalmic solutions on cell cultures. Indian J Ophthalmol [serial online] 1998 [cited 2020 Apr 10];46:37-40. Available from: http://www.ijo.in/text.asp?1998/46/1/37/14979
Viscoelastic ophthalmic solutions available in India are made of either sodium hyaluronate or hydroxy-propylmethylecellulose (HPMC). They are used mainly to maintain anterior chamber and to protect the corneal endothelium and other tissues during cataract surgery. Apart from the several safety criteria these drugs should possess, they should have no inflammatory activity on intraocular use.
In recent times ophthalmic surgeons at the authors' hospital noticed mild but significant intraocular inflammation probably attributable to viscoelastic products used during cataract surgery. Therefore, we studied the in-vitro effects of the available viscoelastic ophthalmic solutions on three continuous tissue culture cell lines for cytotoxicity.
| Materials and Methods|| |
| Viscoelastic ophthalmic solutions|| |
Two pharmaceutical products containing sodium hyaluronate (10 mg/ml) which we designated as A and B, and four pharmaceutical products containing 2% weight by volume of HPMC designated as C, D, E and F were obtained as random samples from our hospital store.
| Tissue culture cell lines|| |
Vero (African green monkey kidney epithelial cell line), HeLa (carcinoma - human cervix), and BHK-21 (baby hamster kidney) cell lines, obtained from National Facility for Animal Tissue Cell Culture, Pune, India and maintained in our tissue culture laboratory, were used. These cell lines were chosen because of the ease with which they are maintained in the laboratory.
| Experimental design|| |
| 96-well tissue culture plate method|| |
Each cell line was subcultured into a 96-well tissue culture (TC) microtitre plate (Tarsons, India) in 100 microlitre volumes per well of cell suspension containing approximately 5 x 105 cells per ml of growth medium of Eagle's minimum essential medium (MEM) (HiMedia, India), supplemented with 10% foetal bovine serum (Biological Industries, Israel). When complete monolayer was obtained (this occurs usually on overnight incubation at 37°C in CO2 atmosphere), the medium from each well was aspirated carefully without disturbing the cells. Using Eagle's MEM growth medium, viscoelastic solutions were diluted to 1:10 and 1:100 dilutions. Ten microlitre volumes of undiluted, 1:10 and 1:100 dilutions of the viscoelastic solutions were placed in quadruplicate wells of each of the cell lines in the 96-well TC microtitre plates. Thus, for the 6 pharmaceutical products, 72 wells (4 wells for each dilution) of the plate were used, and 24 left over wells formed the uninoculated control wells. One hundred microlitres of Eagle's MEM growth medium were placed in the 72 test wells and 110 microlitres of the same were placed in the 24 control wells. An excess of 10 microlitre volumes in control wells was used in place of the same volume of viscoelastic solution used in the test wells. At the end of 72 hours of incubation at 37°C in 10% CO2 atmosphere, the TC plates were examined microscopically. The morphology of the cell lines were studied daily for a period of 72 hours using phase contrast inverted microscope (Diaphot, Nikon, Japan). Any cytopathic (cytotoxic) effect observed was documented. The tissue culture protocols on the distribution of the viscoelastic solutions and the controls in the TC plates were masked from the observer (HNM) who read and documented the results.
After documenting the morphological results, the wells of the TC plate were gently washed three times with phosphate buffered saline (PBS) having pH 7.2. The cells were fixed with cold acetone and stained by 1% crystal violet for a minute. The excess stain was removed by washing 5 times with PBS and the morphology of the cells was further studied for confirmation of the cytotoxicity observed earlier, and any other change in the characteristics of the cells.
| Cover slip tissue culture method|| |
The three cell lines were subcultured in 0.5 ml amounts of cell suspension containing approximately 5 x 105 cells per ml in Eagle's MEM into shell vials containing cover slips. When the monolayer was well formed (usually within 24 hours of incubation at 37°C), the medium was aspirated and cover slip cell cultures were washed with growth medium. Undiluted viscoelastic ophthalmic solutions were inoculated in 10 microlitre volumes into each of the cell lines in cover slip cultures in duplicate shell vials, and 0.5 ml of growth medium was added. Uninoculated control cell lines were also included. At the end of 72 hours the medium was aspirated, the cover slips were gently washed thrice with PBS, cells on the cover slips were fixed in cold acetone and stained with Harris haematoxylin. The stained cover slips were mounted onto microscopic slides and the morphology of cells was examined under bright field binocular microscope (Optiphot, Nikon, Japan) and compared with the controls. The results were documented by photomicrography.
| Microscopic study of viscoelastic ophthalmic solutions|| |
These solutions were examined for the presence of floating particulate materials, using phase contrast inverted microscope (Diaphot, Nikon, Japan). Microscopic slides and cover slips were cleaned thoroughly using tissue paper and examined under the phase contrast microscope, and spotlessly clean spots were chosen. Without removing the microscopic slide, a drop of the ophthalmic solution was placed at these spots carefully using disposable plastic droppers, and spotlessly clean cover slips were placed to cover the solution. The cover slip was pressed with the tip of a forceps to spread the fluid evenly under the cover slip. The solution was carefully studied for floating particles, and if any were found they were documented by photomicrography.
| Results|| |
| Morphology of the cell lines|| |
After overnight incubation, HeLa cell line exposed to the pharmaceutical product D showed rounding and aggregation which increased to a large extent by 72 hours, and this cytopathic (cytotoxic) effect was clearly demonstrable in undiluted as well as in 1:10 dilution [Figure - 1], though this was not seen in 1:100 dilution. The healthy control HeLa cells are shown in [Figure - 2] for comparison. The product D also showed similar cytopathic changes in cover slip cultures of HeLa [Figure - 3] stained with haematoxylin, and its control HeLa cover slip cultures were healthy [Figure - 4]. This product did not show similar effects on Vero and BHK-21 cell lines. Product F showed rounding and aggregation in several areas of the HeLa cell line which was well made out on cover slip cultures [Figure - 5]. None of the other products produced any cytopathic (cytotoxic) effect in the three cell lines tested.
| Microscopic observations|| |
Round refractile and irregularly marginated flat particles of about 20-30 μm in size were found in the product D [Figure - 6]. Dark particles of varying sizes were observed in product F. All other solutions were clear and free of any particulate materials.
| Discussion|| |
This report emphasizes the role of cell culture methods to study the toxicity of viscoelastic ophthalmic solutions. Two pharmaceutical products designated as D and F, both containing HPMC, produced toxicity on HeLa cell line. Clinical studies have shown the protective effect of 1% sodium hyaluronate on corneal endothelial cells.[1-3] But in a sensitive rabbit cell culture in vitro model, Healon appeared to have had toxicity which was attributed to its high viscosity with a high sheer force, or to its formulation in calcium-free vehicle. Glasser et al used 1% sodium hyaluronate, 4% chondroitin sulphate and 2% methylcellulose to study their toxicity in an in-vivo cat model with and without anterior chamber wash out and found that all the solutions provided complete endothelial protection from mechanical trauma but they produced a mild intraocular inflammation at one and two days after anterior chamber instillation. The above solutions in various concentrations and combinations showed toxicity to cultured corneal endothelial cells depending on the vehicle and ionic composition. Since many viscoelastic preparations are without calcium or magnesium (because they cause precipitation), loss of these cations may cause toxicity. But this theory does not hold good in our in-vitro cell culture model because both these ions are available in the tissue culture growth medium used, and further, the products D and F showing toxicity on HeLa cell line did not have any such effect on Vero and BHK-21 cell lines which are kidney epithelial cells. Whether this specific cytotoxic effect is likely to be more pronounced on cell line derived from carcinoma cells than on kidney cells is hypothetical.
Simple phase contrast microscopy has demonstrated the floating particles in the pharmaceutical products D and F. Whether these floating particles have a role in the cytotoxicity can only be conjectured since the other two cell lines have not been affected. Rosen et al examined 6 HPMC ophthalmic solutions (3 from United Kingdom, 2 from West Germany, and 1 from Swedish Hospital Pharmacy) and found a variety of particulate debris of botanical origin in varying amounts. They also found glass fragments in one of the UK preparations, and they concluded that the methods used to purify the product were ineffective and inadequate.
We conclude that HeLa cell line could be used for cytotoxicity studies of viscoelastic ophthalmic solutions. We may hypothesise that the cytotoxicity of the products D and F clearly demonstrable in HeLa cell line could possibly reflect in the form of intraocular inflammation atleast in some susceptible patients. We would like to recommend that toxicity of pharmaceutic products in general can be tested in commonly available and easily maintained tissue culture cell lines as we generally do for bacterial toxins. It is essential that there should be a method to verify the presence of floating particles in the products before they are put into use for cataract surgery.
| References|| |
Pape LG, Balazs EA. The use of sodium hyaluronate (Healon) in human anterior segment surgery. Ophthalmology
Holmberg AS, Philipson BT. Sodium hyaluronate in cataract surgery. I. Report on the use of Healon in two different types of intracapsular cataract surgery. Ophthalmology
Holmberg AS, Philipson BT. Sodium hyaluronate in cataract surgery. II. Report on the use of Healon in extracapsular cataract surgery using phacoemulsification. Ophthalmology
Liesegang TJ. Viscoelastic substances in ophthalmology:therapeutic review. Surv Ophthalmol
Glasser DB, Matsuda M, Edelhauser HF. A comparison of the efficacy and toxicity of and intraocular pressure response to viscous solutions in the anterior chamber. Arch Ophthalmol
McCully JP, Stern ME, Meyer DR. Assessment of the comparative toxicity of viscoelastic substances. Invest Ophthalmol Vis Sci
Rosen ES, Gregory RPF, Barnett F. Is 2% hydroxypropylmethylecellulose a safe solution for intraoperative clinical applications? J Cat Ref Surg
[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6]