Primary culture of rat lens fibers and the role of tonicity of the medium in calcium-induced globulization

L. F. Wang, A. Bhatnagar, Naseem Ansari, Satish Srivastava

Research output: Contribution to journalArticle

Abstract

Purpose. To maintain rat lens fibers in culture and to investigate the role of cell swelling in the distintegrative globulization of fibers. Methods. Viable and morphologically intact fibers were isolated from rat lens cortex and cultured at 37°C in HEPES buffered (pH 7.4), isotonic sucrose solution containing a combination of additives such as trypsin inhibitor, fetal calf serum, EDTA, GSH, and vitamins. The effects of extracellular ions and tonicity on fiber cell globulization were investigated. Results. Single fibers cultured for 5 days in HEPES-isotonic sucrose solution containing EDTA, 10 mM, trypsin inhibitor, 10 μg/ml, and GSH, 10 mM, remained viable (as judged by trypan blue exclusion) and maintained fiber cell morphology. Decrease of sucrose solution's tonicity to 212 mOsm (hypotonic) caused globulization of fiber cells; globulization time (tg) was 75± 7 min (n=30), whereas in Ringer's solution with 1 mM calcium tg was 33 ± 1 min (n=24). Exposure of fiber cells to Ringer's solution for 5 min, followed by superfusion with either isotonic, hypertonic (400 mOsm), or hypotonic HEPES-sucrose resulted in tg of 108± 5, 99±3, and 51±6 min (n=20 to 40), respectively. Similar results were obtained with initial 5 min exposure of fiber cells to Ringer's containing 10-8 M Ca2+. Conclusions. Addition of trypsin inhibitor, metal chelator and GSH in the culture media preserved the viability of isolated lens fibers for 5 days. A brief exposure to ionic medium commits isolated fibers to globulize; globulization is accelerated by decreasing medium tonicity. These results indicate that the ion-regulating mechanism of cortical fibers is different from lens epithelium.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

Fingerprint

Lenses
HEPES
Calcium
Trypsin Inhibitors
Sucrose
Isotonic Solutions
Edetic Acid
Ions
Trypan Blue
Chelating Agents
Vitamins
Culture Media
Epithelium
Metals
Serum
Ringer's solution

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Primary culture of rat lens fibers and the role of tonicity of the medium in calcium-induced globulization. / Wang, L. F.; Bhatnagar, A.; Ansari, Naseem; Srivastava, Satish.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

@article{44900776065347388dbe07b1f7bd16f4,
title = "Primary culture of rat lens fibers and the role of tonicity of the medium in calcium-induced globulization",
abstract = "Purpose. To maintain rat lens fibers in culture and to investigate the role of cell swelling in the distintegrative globulization of fibers. Methods. Viable and morphologically intact fibers were isolated from rat lens cortex and cultured at 37°C in HEPES buffered (pH 7.4), isotonic sucrose solution containing a combination of additives such as trypsin inhibitor, fetal calf serum, EDTA, GSH, and vitamins. The effects of extracellular ions and tonicity on fiber cell globulization were investigated. Results. Single fibers cultured for 5 days in HEPES-isotonic sucrose solution containing EDTA, 10 mM, trypsin inhibitor, 10 μg/ml, and GSH, 10 mM, remained viable (as judged by trypan blue exclusion) and maintained fiber cell morphology. Decrease of sucrose solution's tonicity to 212 mOsm (hypotonic) caused globulization of fiber cells; globulization time (tg) was 75± 7 min (n=30), whereas in Ringer's solution with 1 mM calcium tg was 33 ± 1 min (n=24). Exposure of fiber cells to Ringer's solution for 5 min, followed by superfusion with either isotonic, hypertonic (400 mOsm), or hypotonic HEPES-sucrose resulted in tg of 108± 5, 99±3, and 51±6 min (n=20 to 40), respectively. Similar results were obtained with initial 5 min exposure of fiber cells to Ringer's containing 10-8 M Ca2+. Conclusions. Addition of trypsin inhibitor, metal chelator and GSH in the culture media preserved the viability of isolated lens fibers for 5 days. A brief exposure to ionic medium commits isolated fibers to globulize; globulization is accelerated by decreasing medium tonicity. These results indicate that the ion-regulating mechanism of cortical fibers is different from lens epithelium.",
author = "Wang, {L. F.} and A. Bhatnagar and Naseem Ansari and Satish Srivastava",
year = "1996",
month = "2",
day = "15",
language = "English (US)",
volume = "37",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "3",

}

TY - JOUR

T1 - Primary culture of rat lens fibers and the role of tonicity of the medium in calcium-induced globulization

AU - Wang, L. F.

AU - Bhatnagar, A.

AU - Ansari, Naseem

AU - Srivastava, Satish

PY - 1996/2/15

Y1 - 1996/2/15

N2 - Purpose. To maintain rat lens fibers in culture and to investigate the role of cell swelling in the distintegrative globulization of fibers. Methods. Viable and morphologically intact fibers were isolated from rat lens cortex and cultured at 37°C in HEPES buffered (pH 7.4), isotonic sucrose solution containing a combination of additives such as trypsin inhibitor, fetal calf serum, EDTA, GSH, and vitamins. The effects of extracellular ions and tonicity on fiber cell globulization were investigated. Results. Single fibers cultured for 5 days in HEPES-isotonic sucrose solution containing EDTA, 10 mM, trypsin inhibitor, 10 μg/ml, and GSH, 10 mM, remained viable (as judged by trypan blue exclusion) and maintained fiber cell morphology. Decrease of sucrose solution's tonicity to 212 mOsm (hypotonic) caused globulization of fiber cells; globulization time (tg) was 75± 7 min (n=30), whereas in Ringer's solution with 1 mM calcium tg was 33 ± 1 min (n=24). Exposure of fiber cells to Ringer's solution for 5 min, followed by superfusion with either isotonic, hypertonic (400 mOsm), or hypotonic HEPES-sucrose resulted in tg of 108± 5, 99±3, and 51±6 min (n=20 to 40), respectively. Similar results were obtained with initial 5 min exposure of fiber cells to Ringer's containing 10-8 M Ca2+. Conclusions. Addition of trypsin inhibitor, metal chelator and GSH in the culture media preserved the viability of isolated lens fibers for 5 days. A brief exposure to ionic medium commits isolated fibers to globulize; globulization is accelerated by decreasing medium tonicity. These results indicate that the ion-regulating mechanism of cortical fibers is different from lens epithelium.

AB - Purpose. To maintain rat lens fibers in culture and to investigate the role of cell swelling in the distintegrative globulization of fibers. Methods. Viable and morphologically intact fibers were isolated from rat lens cortex and cultured at 37°C in HEPES buffered (pH 7.4), isotonic sucrose solution containing a combination of additives such as trypsin inhibitor, fetal calf serum, EDTA, GSH, and vitamins. The effects of extracellular ions and tonicity on fiber cell globulization were investigated. Results. Single fibers cultured for 5 days in HEPES-isotonic sucrose solution containing EDTA, 10 mM, trypsin inhibitor, 10 μg/ml, and GSH, 10 mM, remained viable (as judged by trypan blue exclusion) and maintained fiber cell morphology. Decrease of sucrose solution's tonicity to 212 mOsm (hypotonic) caused globulization of fiber cells; globulization time (tg) was 75± 7 min (n=30), whereas in Ringer's solution with 1 mM calcium tg was 33 ± 1 min (n=24). Exposure of fiber cells to Ringer's solution for 5 min, followed by superfusion with either isotonic, hypertonic (400 mOsm), or hypotonic HEPES-sucrose resulted in tg of 108± 5, 99±3, and 51±6 min (n=20 to 40), respectively. Similar results were obtained with initial 5 min exposure of fiber cells to Ringer's containing 10-8 M Ca2+. Conclusions. Addition of trypsin inhibitor, metal chelator and GSH in the culture media preserved the viability of isolated lens fibers for 5 days. A brief exposure to ionic medium commits isolated fibers to globulize; globulization is accelerated by decreasing medium tonicity. These results indicate that the ion-regulating mechanism of cortical fibers is different from lens epithelium.

UR - http://www.scopus.com/inward/record.url?scp=33750186802&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33750186802&partnerID=8YFLogxK

M3 - Article

VL - 37

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 3

ER -