Comparison of viability of oral epithelial cells stored by different freezing methods

Do-Young Baek; Seung-Jong Lee; Han-Sung Jung; EuiSeong Kim

doi:10.5395/JKACD.2009.34.6.491

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Original Article Comparison of viability of oral epithelial cells stored by different freezing methods: Do-Young Baek, Seung-Jong Lee, Han-Sung Jung, EuiSeong Kim; 2009;34(6):-499.
DOI: https://doi.org/10.5395/JKACD.2009.34.6.491
Published online: November 30, 2009

Department of Conservative Dentistry, College of Dentistry, Yonsei University, Korea.

Corresponding Author: Euiseong Kim. Department of Conservative Dentistry, College of Dentistry, Yonsei University, 134 Shinchon-dong, Sudaemoon-gu, Seoul, Korea. Tel: 82-2-2228-3151, Fax: 82-2-313-7575, andyendo@yuhs.ac

• Received: September 1, 2009 • Revised: September 25, 2009 • Accepted: October 6, 2009

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Abstract
REFERENCES

Abstract

This study examined the influence of the storage methods on the viability of oral epithelial cells using conventional cell freezing storage, slow freezing preservation, rapid freezing preservation, and slow freezing preservation with a pressure of 2 Mpa or 3 Mpa. The cell viability was evaluated by cell counting, WST-1 and the clonogenic capacity after 6 days of freezing storage. After 6 days, the frozen cells were thawed rapidly, and the cell counting, WST-1, and clonogenic capacity values were measured and compared.
1. The results from cell counting demonstrated that conventional cryopreservation, slow freezing under a 2 Mpa pressure and slow freezing under a 3 Mpa pressure showed significantly higher values than slow freezing preservation and rapid freezing preservation (p<0.05).
2. The results from the optical density by WST-1 demonstrated that slow freezing under a 2 Mpa pressure showed significantly higher values than slow freezing preservation and rapid freezing preservation (p<0.05).
3. The clonogenic capacity demonstrated that slow freezing under a 2 Mpa pressure showed significantly higher values than slow freezing preservation and rapid freezing preservation (p<0.05).
Keywords: WST-1; Cell counting; Clonogenic capacity; Slow freezing under pressure; Slow freezing; Rapid freezing

Figure 1

1℃/min freezing container "Mr. Frosty"

Figure 2

Schematic diagram of program freezer with pressure vessel.

a. Oxygen container : 2,3 Mpa of pressure

b. Program freezer

c. Pressure bottle

d. 2ml Cryotube: 1ml 65%RPMI+30%FBS+5%DMSO

e. Cell suspension

f. Pressure valve

g. Thermometer

Figure 3

Cell counting by hemacytometer slide and trypan blue

Figure 4

Standard curve of WST-1 using monolayer epithelial cell(YD-38)

Figure 5

Clonogenic capacity of experimental groups

Figure 6

Schematic of physical events in cells during freezing.

Table 1

The averages and standard deviations of viable cell number (logN)

a,b: Different letters denote statistically significant (p<0.05)

Table 2

The Median and IQR of optical density of WST-1

a,b,c: Different letters denote statistically significant (p<0.05)

Table 3

The Median and IQR of the number of colonies (number of cells seeded : 1×10³)

a,b,c: Different letters denote statistically significant (p<0.05)

REFERENCES

1. Kim JW, Kim US, Kim J, Lee SJ. Evaluation of Periodontal Ligament Cell Viability in Rat Teeth after Frozen Preservation using in-vivo MTT Assay. J Korean Acad Conserv Dent. 2006;31: 192-202.Article
2. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol. 1984;247: C125-C142.Article PubMed
3. Farrant J. Water transport and cell survival in cryobiological procedures. Philos Trans R Soc Lond B Biol Sci. 1977;278(959):191-205.Article PubMed PDF
4. Fujikawa S. "Freeze-fracture and etching studies on membrane damage on human erythrocytes caused by formation of intracellular ice.". Cryobiology. 1980;17(4):351-362.Article PubMed
5. Diller KR, Cravalho EG. A cryomicroscope for the study of freezing and thawing processes in biological cells. Cryobiology. 1970;7(4):191-199.Article PubMed
6. Kawata T. Tooth transplantation by teeth bank-approach to human-Hiroshima. 2005;Department of Orthodontics, Hiroshima University School of Dentistry.
7. Ahn HJ, Kim US, Kim J, Kim DW, Kim KY, Lee CY, Lee SJ. Evaluation of the Viability of Periodontal Ligament Cell in Rat Teeth using Slow Cryopreservation Method with Magnetic Field. J Korean Acad Conserv Dent. 2008;33: 332-340.Article
8. Pribenszky C, Molnar M, Cseh S, Solti L. Improving post-thaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge. Anim Reprod Sci. 2005;87(1-2):143-150.Article PubMed
9. Pribenszky C, Molnar M, Ulrich P, et al. Pressure assisted cryopreservation. Reprod Domest Anim. 2005;40: 228-344.PubMed
10. Kuo YH, Pribenszky Cs, Huang SY. Higher litter size is achieved by the insemination of high hydrostatic pressure-treated frozen-thawed boar semen. Theriogenology. 2008;70: 1395-1396.Article
11. Berridge MV, Tan AS, McCoy KD, Wang R. The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica (Indianapolis). 1996;4: 15-20.
12. Alotto D, Ariotti S, Graziano S, Verrua R, Stella M, Magliacani G, Castagnoli C. The role of quality control in a skin bank: tissue viability determination. Cell Tissue Bank. 2002;3(1):3-10.PubMed
13. Czochrowska EM, Stenvik A, et al. "Outcome of tooth transplantation: survival and success rates 17-41 years posttreatment.". Am J Orthod Dentofacial Orthop. 2002;121(2):110-119 quiz 193.Article PubMed
14. Jonsson T, Sigurdsson TJ. "Autotransplantation of premolars to premolar sites. A long-term follow-up study of 40 consecutive patients.". Am J Orthod Dentofacial Orthop. 2004;125(6):668-675.Article PubMed
15. Mazur P. "Equilibrium, quasi-equilibrium, and non-equilibrium freezing of mammalian embryos.". Cell Biophys. 1990;17(1):53-92.Article PubMed PDF
16. Mazur P, Leibo SP, Chu EH. A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells. Exp Cell Res. 1972;71(2):345-355.PubMed
17. Rapatz G, Sullivan JJ, et al. "Preservation of erythrocytes in blood containing various cryoprotective agents, frozen at various rates and brought to a given final temperature.". Cryobiology. 1968;5(1):18-25.Article PubMed
18. Thorpe PE, Knight SC, et al. "Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques.". Cryobiology. 1976;13(2):126-133.Article PubMed
19. Leibo SP, McGrath JJ, et al. "Microscopic observation of intracellular ice formation in unfertilized mouse ova as a function of cooling rate.". Cryobiology. 1978;15(3):257-271.Article PubMed
20. Abe F, Kato C, Horikoshi K. Pressure regulated metabolism in microorganisms. Trends Microbiol. 1999;7(11):447-453.Article PubMed
21. Aldridge BE, Bruner LJ. Pressure effects on mechanisms of charge transport across bilayer membranes. Biochem Biophys Acta. 1985;817(2):343-354.Article PubMed
22. Huang SY, Pribenszky C, Kuo YH, et al. The effect of hydrostatic pressure treatment on the protein profile of boar spermatozoa before and after freezing. Theriogenology. 2008;70: 1391.Article
23. Parsell DA, Lindquist S. The function of heat shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Ann Rev Genet. 1993;27: 437-496.Article PubMed
24. Pelham HR. Heat shock proteins: coming in from cold. Nature. 1988;332: 776-777.Article PubMed PDF
25. Du Y, Lin L, Schmidt M, Bogh IB, Kragh PM, Sorensen CB, Li J, Purup S, Pribenszky C, Molnar M, Kuwayama M, Zhang X, Yang H, Bolund L, Vajta G. High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival. Cloning Stem Cells. 2008;10(3):325-330.Article PubMed
26. Lee YE, Kim US, Kim J, Han SH, Lee SJ. The Efficacy of Programmed Cryo-preservation under Pressure in Rat Periodontal Ligament Cells. J Korean Acad Conserv Dent. 2009;34: 356-363.Article

Tables & Figures

Figure 1

1℃/min freezing container "Mr. Frosty"

Figure 2

Schematic diagram of program freezer with pressure vessel.

a. Oxygen container : 2,3 Mpa of pressure

b. Program freezer

c. Pressure bottle

d. 2ml Cryotube: 1ml 65%RPMI+30%FBS+5%DMSO

e. Cell suspension

f. Pressure valve

g. Thermometer

Figure 3

Cell counting by hemacytometer slide and trypan blue

Figure 4

Standard curve of WST-1 using monolayer epithelial cell(YD-38)

Figure 5

Clonogenic capacity of experimental groups

Figure 6

Schematic of physical events in cells during freezing.

Table 1

The averages and standard deviations of viable cell number (logN)

a,b: Different letters denote statistically significant (p<0.05)

Table 2

The Median and IQR of optical density of WST-1

a,b,c: Different letters denote statistically significant (p<0.05)

Table 3

The Median and IQR of the number of colonies (number of cells seeded : 1×10³)

a,b,c: Different letters denote statistically significant (p<0.05)

REFERENCES

1. Kim JW, Kim US, Kim J, Lee SJ. Evaluation of Periodontal Ligament Cell Viability in Rat Teeth after Frozen Preservation using in-vivo MTT Assay. J Korean Acad Conserv Dent. 2006;31: 192-202.Article
2. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol. 1984;247: C125-C142.Article PubMed
3. Farrant J. Water transport and cell survival in cryobiological procedures. Philos Trans R Soc Lond B Biol Sci. 1977;278(959):191-205.Article PubMed PDF
4. Fujikawa S. "Freeze-fracture and etching studies on membrane damage on human erythrocytes caused by formation of intracellular ice.". Cryobiology. 1980;17(4):351-362.Article PubMed
5. Diller KR, Cravalho EG. A cryomicroscope for the study of freezing and thawing processes in biological cells. Cryobiology. 1970;7(4):191-199.Article PubMed
6. Kawata T. Tooth transplantation by teeth bank-approach to human-Hiroshima. 2005;Department of Orthodontics, Hiroshima University School of Dentistry.
7. Ahn HJ, Kim US, Kim J, Kim DW, Kim KY, Lee CY, Lee SJ. Evaluation of the Viability of Periodontal Ligament Cell in Rat Teeth using Slow Cryopreservation Method with Magnetic Field. J Korean Acad Conserv Dent. 2008;33: 332-340.Article
8. Pribenszky C, Molnar M, Cseh S, Solti L. Improving post-thaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge. Anim Reprod Sci. 2005;87(1-2):143-150.Article PubMed
9. Pribenszky C, Molnar M, Ulrich P, et al. Pressure assisted cryopreservation. Reprod Domest Anim. 2005;40: 228-344.PubMed
10. Kuo YH, Pribenszky Cs, Huang SY. Higher litter size is achieved by the insemination of high hydrostatic pressure-treated frozen-thawed boar semen. Theriogenology. 2008;70: 1395-1396.Article
11. Berridge MV, Tan AS, McCoy KD, Wang R. The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica (Indianapolis). 1996;4: 15-20.
12. Alotto D, Ariotti S, Graziano S, Verrua R, Stella M, Magliacani G, Castagnoli C. The role of quality control in a skin bank: tissue viability determination. Cell Tissue Bank. 2002;3(1):3-10.PubMed
13. Czochrowska EM, Stenvik A, et al. "Outcome of tooth transplantation: survival and success rates 17-41 years posttreatment.". Am J Orthod Dentofacial Orthop. 2002;121(2):110-119 quiz 193.Article PubMed
14. Jonsson T, Sigurdsson TJ. "Autotransplantation of premolars to premolar sites. A long-term follow-up study of 40 consecutive patients.". Am J Orthod Dentofacial Orthop. 2004;125(6):668-675.Article PubMed
15. Mazur P. "Equilibrium, quasi-equilibrium, and non-equilibrium freezing of mammalian embryos.". Cell Biophys. 1990;17(1):53-92.Article PubMed PDF
16. Mazur P, Leibo SP, Chu EH. A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells. Exp Cell Res. 1972;71(2):345-355.PubMed
17. Rapatz G, Sullivan JJ, et al. "Preservation of erythrocytes in blood containing various cryoprotective agents, frozen at various rates and brought to a given final temperature.". Cryobiology. 1968;5(1):18-25.Article PubMed
18. Thorpe PE, Knight SC, et al. "Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques.". Cryobiology. 1976;13(2):126-133.Article PubMed
19. Leibo SP, McGrath JJ, et al. "Microscopic observation of intracellular ice formation in unfertilized mouse ova as a function of cooling rate.". Cryobiology. 1978;15(3):257-271.Article PubMed
20. Abe F, Kato C, Horikoshi K. Pressure regulated metabolism in microorganisms. Trends Microbiol. 1999;7(11):447-453.Article PubMed
21. Aldridge BE, Bruner LJ. Pressure effects on mechanisms of charge transport across bilayer membranes. Biochem Biophys Acta. 1985;817(2):343-354.Article PubMed
22. Huang SY, Pribenszky C, Kuo YH, et al. The effect of hydrostatic pressure treatment on the protein profile of boar spermatozoa before and after freezing. Theriogenology. 2008;70: 1391.Article
23. Parsell DA, Lindquist S. The function of heat shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Ann Rev Genet. 1993;27: 437-496.Article PubMed
24. Pelham HR. Heat shock proteins: coming in from cold. Nature. 1988;332: 776-777.Article PubMed PDF
25. Du Y, Lin L, Schmidt M, Bogh IB, Kragh PM, Sorensen CB, Li J, Purup S, Pribenszky C, Molnar M, Kuwayama M, Zhang X, Yang H, Bolund L, Vajta G. High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival. Cloning Stem Cells. 2008;10(3):325-330.Article PubMed
26. Lee YE, Kim US, Kim J, Han SH, Lee SJ. The Efficacy of Programmed Cryo-preservation under Pressure in Rat Periodontal Ligament Cells. J Korean Acad Conserv Dent. 2009;34: 356-363.Article

Citations

Citations to this article as recorded by

Evaluation of the Viability of Rat Periodontal Ligament Cells after Storing at 0℃/2 MPa Condition up to One Week: In Vivo MTT Method
Sun Mi Jang, Sin-Yeon Cho, Eui-Seong Kim, Il-Young Jung, Seung Jong Lee
Journal of Korean Dental Science.2016; 9(1): 1. CrossRef
The evaluation of periodontal ligament cells of rat teeth after low-temperature preservation under high pressure
Jin-Ho Chung, Jin Kim, Seong-Ho Choi, Eui-Seong Kim, Jiyong Park, Seung-Jong Lee
Journal of Korean Academy of Conservative Dentistry.2010; 35(4): 285. CrossRef

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Comparison of viability of oral epithelial cells stored by different freezing methods

J Korean Acad Conserv Dent. 2009;34(6):491-499. Published online November 30, 2009

DOI: https://doi.org/10.5395/JKACD.2009.34.6.491

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Figure

Comparison of viability of oral epithelial cells stored by different freezing methods

Figure 1 1℃/min freezing container "Mr. Frosty"

Figure 2 Schematic diagram of program freezer with pressure vessel. a. Oxygen container : 2,3 Mpa of pressure b. Program freezer c. Pressure bottle d. 2ml Cryotube: 1ml 65%RPMI+30%FBS+5%DMSO e. Cell suspension f. Pressure valve g. Thermometer

Figure 3 Cell counting by hemacytometer slide and trypan blue

Figure 4 Standard curve of WST-1 using monolayer epithelial cell(YD-38)

Figure 5 Clonogenic capacity of experimental groups

Figure 6 Schematic of physical events in cells during freezing.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Comparison of viability of oral epithelial cells stored by different freezing methods

Table 1

The averages and standard deviations of viable cell number (logN)

a,b: Different letters denote statistically significant (p<0.05)

Table 2

The Median and IQR of optical density of WST-1

a,b,c: Different letters denote statistically significant (p<0.05)

Table 3

The Median and IQR of the number of colonies (number of cells seeded : 1×10³)

a,b,c: Different letters denote statistically significant (p<0.05)

Table 1 The averages and standard deviations of viable cell number (logN)

a,b: Different letters denote statistically significant (p<0.05)

Table 2 The Median and IQR of optical density of WST-1

a,b,c: Different letters denote statistically significant (p<0.05)

Table 3 The Median and IQR of the number of colonies (number of cells seeded : 1×103)

a,b,c: Different letters denote statistically significant (p<0.05)