Skip to main content

Advertisement

SpringerLink
Log in

An optimised protocol for the production of autologous serum eyedrops

  • Laboratory Investigation
  • Published:
Graefe's Archive for Clinical and Experimental Ophthalmology Aims and scope Submit manuscript

Abstract

Background

Serum eyedrops have been successfully used in the treatment of severe dry eye, persistent epithelial defects and other severe ocular surface disorders. A number of clinical studies showed a variable efficacy of this approach, but the parameters for the production of this blood product varied significantly. In order to establish an optimised protocol for the production of serum eyedrops, we examined the effect of various clotting times, centrifugation forces, types of diluent and dilutions on the concentration of growth factors, fibronectin, and vitamins in serum and tested the epitheliotrophic capacity of these serum modifications in a cell culture model of human SV-40-immortalised corneal epithelial cells (HCE-T).

Methods

Serum samples were prepared with a clotting time of 20, 60 or 120 min, a centrifugation force of 500×g or 3,000×g, and diluted with BSS or isotonic saline. The concentrations of EGF, TGF-β1, PDGF-AB, FGF, HGF, fibronectin, vitamin A and vitamin E in these samples were evaluated with ELISA and HPLC. HCE-T cells were incubated for 24, 48, 72, 96 and 144 h with 100, 50, 25, 12.5, 6.25 and 3.125% serum in diluent, and cell proliferation, migration and differentiation were evaluated by means of a luminescence-based ATP assay, a colony-dispersion assay and scanning electron microscopy.

Results

Using a longer clotting time resulted in an increased concentration of all the epitheliotrophic factors examined in serum; the diffference was statistically significant for EGF, TGF-β1 and HGF. Increasing the g force of centrifugation from 500×g to 3,000×g resulted in significantly less TGF-β1, but more EGF and vitamin A. Cell proliferation was better supported by serum prepared with 3,000×g and diluted with BSS. Serum prepared with a longer clotting time yielded better cell migration and differentiation.

Conclusion

Clotting time, centrifugation and diluents have a significant impact on the composition and epitheliotrophic effects of serum. A long clotting time (≥120 min), a sharp centrifugation (3,000×g for 15 min) and dilution with BSS improve the ability of serum eyedrops to support proliferation, migration and differentiation of corneal epithelial cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Andresen JL, Ledet T, Ehlers N (1997) Keratocyte migration and peptide growth factors: the effect of PDGF, bFGF, EGF, IGF-I, aFGF and TGF-beta on human keratocyte migration in a collagen gel. Curr Eye Res 16:605–613

    Google Scholar 

  2. Araki-Sasaki K, Ohashi Y, Sasabe T, Hayashi K, Watanabe H, Tano Y, Handa H (1995) An SV40-immortalized human corneal epithelial cell line and its characterization. Invest Ophthalmol Vis Sci 36:614–621

    Google Scholar 

  3. Bilgihan K, Adiguzel U, Sezer C, Akyol G, Hasanreisoglu B (2001) Effects of topical vitamin E on keratocyte apoptosis after traditional photorefractive keratectomy. Ophthalmologica 215:192–196

    Google Scholar 

  4. Chandrasekher G, Kakazu AH, Bazan HE (2001) HGF- and KGF-induced activation of PI-3K/p70 s6 kinase pathway in corneal epithelial cells: its relevance in wound healing. Exp Eye Res 73:191–202

    Google Scholar 

  5. Collins MK, Perkins GR, Rodriguez-Tarduchy G, Nieto MA, Lopez-Rivas A (1994) Growth factors as survival factors: regulation of apoptosis. Bioessays 16:133–138

    Google Scholar 

  6. del Castillo JM, de la Casa JM, Sardina RC, Fernandez RM, Feijoo JG, Gomez AC, Rodero MM, Sanchez JG (2002) Treatment of recurrent corneal erosions using autologous serum. Cornea 21:781–783

    Google Scholar 

  7. Dugrillon A, Lauber S, Nguyen X, Klueter H (eds) (2002) Platelets applied to wounds and in tissue regeneration: induction of proliferation apoptosis by platelet membranes

    Google Scholar 

  8. Er H, Uzmez E (1998) Effects of transforming growth factor-beta 2, interleukin 6 and fibronectin on corneal epithelial wound healing. Eur J Ophthalmol 8:224–229

    Google Scholar 

  9. Ferreira de Souza R, Kruse FE, Seitz B (2001) Autologous serum for otherwise therapy resistant corneal epithelial defects—prospective report on the first 70 eyes. Klin Monatsbl Augenheilkd 218:720–726

    Google Scholar 

  10. Fox RI, Chan R, Mishelson JB (1984) Beneficial effect of artificial tears made with autologous serum in patients with keratoconjunctivitis sicca. Arthritis Rheum 27:459–461

    Google Scholar 

  11. Garlick JA, Taichman LB (1994) Fate of human keratinocytes during reepithelialization in an organotypic culture model. Lab Invest 70:916–924

    Google Scholar 

  12. Geerling G, Sieg P, Bastian GO, Laqua H (1998) Transplantation of the autologous submandibular gland for most severe cases of keratoconjunctivitis sicca. Ophthalmology 105:327–335

    Google Scholar 

  13. Geerling G, Daniels JT, Dart JK, Cree IA, Khaw PT (2001) Toxicity of natural tear substitutes in a fully defined culture model of human corneal epithelial cells. Invest Ophthalmol Vis Sci 42:948–956

    Google Scholar 

  14. Goto E, Shimmura S, Shimazaki J, Tsubota K (2001) Treatment of superior limbic keratoconjunctivitis by application of autologous serum. Cornea 20:807–810

    Google Scholar 

  15. Grant MB, Khaw PT, Schultz GS, Adams JL, Shimizu RW (1992) Effects of epidermal growth factor, fibroblast growth factor, and transforming growth factor-beta on corneal cell chemotaxis. Invest Ophthalmol Vis Sci 33:3292–3301

    Google Scholar 

  16. Haber M, Cao Z, Panjwani N, Bedenice D, Li WW, Provost PJ (2003) Effects of growth factors (EGF, PDGF-BB and TGF-beta 1) on cultured equine epithelial cells and keratocytes: implications for wound healing. Vet Ophthalmol 6:211–217

    Google Scholar 

  17. Kay EP, Lee MS, Seong GJ, Lee YG (1998) TGF-beta s stimulate cell proliferation via an autocrine production of FGF-2 in corneal stromal fibroblasts. Curr Eye Res 17:286–293

    Google Scholar 

  18. Kim JM, Stapleton F, Willcox MD (1999) Induction of apoptosis in human corneal epithelial cells in vitro. Aust N Z J Ophthalmol 27:214–217

    Google Scholar 

  19. Kim WJ, Mohan RR, Wilson SE (1999) Effect of PDGF, IL-1alpha, and BMP2/4 on corneal fibroblast chemotaxis: expression of the platelet-derived growth factor system in the cornea. Invest Ophthalmol Vis Sci 40:1364–1372

    Google Scholar 

  20. Kitazawa T, Kinoshita S, Fujita K, Araki K, Watanabe H, Ohashi Y, Manabe R (1990) The mechanism of accelerated corneal epithelial healing by human epidermal growth factor. Invest Ophthalmol Vis Sci 31:1773–1778

    Google Scholar 

  21. Li Q, Weng J, Mohan RR, Bennett GL, Schwall R, Wang ZF, Tabor K, Kim J, Hargrave S, Cuevas KH, Wilson SE (1996) Hepatocyte growth factor and hepatocyte growth factor receptor in the lacrimal gland, tears, and cornea. Invest Ophthalmol Vis Sci 37:727–739

    Google Scholar 

  22. McDonnell PJ, Schanzlin DJ, Rao NA (1988) Immunoglobulin deposition in the cornea after application of autologous serum. Arch Ophthalmol 106:1423–1425

    Google Scholar 

  23. Mishima H, Nakamura M, Murakami J, Nishida T, Otori T (1992) Transforming growth factor-beta modulates effects of epidermal growth factor on corneal epithelial cells. Curr Eye Res 11:691–696

    Google Scholar 

  24. Noble B, Loh R, MacLennan S, Pesudovs K, Reynolds A, Bridges L, Burr J, Stewart O, Quereshi S (2004) Comparison of autologous serum eye drops with conventional therapy in a randomised crossover trial for ocular surface disease. Br J Ophthalmol 88:647-652

    Google Scholar 

  25. Noecker R (2001) Effects of common ophthalmic preservatives on ocular health. Adv Ther 18:205–215

    Google Scholar 

  26. Ogawa Y, Okamoto S, Mori T, Yamada M, Mashima Y, Watanabe R, Kuwana M, Tsubota K, Ikeda Y, Oguchi Y (2003) Autologous serum eye drops for the treatment of severe dry eye in patients with chronic graft-versus-host disease. Bone Marrow Transplant 31:579–583

    Google Scholar 

  27. Pfister RR, Renner ME (1978) The corneal and conjunctival surface in vitamin A deficiency: a scanning electron microscopy study. Invest Ophthalmol Vis Sci 17:874–883

    Google Scholar 

  28. Pilcher BK, Dumin JA, Sudbeck BD, Krane SM, Welgus HG, Parks WC (1997) The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix. J Cell Biol 137:1445–1457

    Google Scholar 

  29. Poon AC, Geerling G, Dart JK, Fraenkel GE, Daniels JT (2001) Autologous serum eyedrops for dry eyes and epithelial defects: clinical and in vitro toxicity studies. Br J Ophthalmol 85:1188–1197

    CAS  PubMed  Google Scholar 

  30. Rao RC, Varani J, Soong HK (1992) FGF promotes corneal stromal fibroblast motility. J Ocul Pharmacol 8:77–81

    Google Scholar 

  31. Rocha EM, Pelegrino FS, de Paiva CS, Vigorito AC, de Souza CA (2000) GVHD dry eyes treated with autologous serum tears. Bone Marrow Transplant 25:1101–1103

    Google Scholar 

  32. Rocha EM, Cunha DA, Carneiro EM, Boschero AC, Saad MJ, Velloso LA (2002) Insulin, insulin receptor and insulin-like growth factor-I receptor on the human ocular surface. Adv Exp Med Biol 506:607–610

    Google Scholar 

  33. Rodeck U, Jost M, Kari C, Shih DT, Lavker RM, Ewert DL, Jensen PJ (1997) EGF-R dependent regulation of keratinocyte survival. J Cell Sci 110 (Pt 2):113–121

    Google Scholar 

  34. Sommer A (1998) Xerophthalmia and vitamin A status. Prog Retin Eye Res 17:9–31

    Google Scholar 

  35. Spigelman AV, Deutsch TA, Sugar J (1987) Application of homologous fibronectin to persistent human corneal epithelial defects. Cornea 6:128–130

    Google Scholar 

  36. Tananuvat N, Daniell M, Sullivan LJ, Yi Q, McKelvie P, McCarty DJ, Taylor HR (2001) Controlled study of the use of autologous serum in dry eye patients. Cornea 20:802–806

    Google Scholar 

  37. Tripathi BJ, Tripathi RC (1989) Cytotoxic effects of benzalkonium chloride and chlorobutanol on human corneal epithelial cells in vitro. Lens Eye Toxic Res 6:395–403

    Google Scholar 

  38. Tsubota K, Satake Y, Ohyama M, Toda I, Takano Y, Ono M, Shinozaki N, Shimazaki J (1996) Surgical reconstruction of the ocular surface in advanced ocular cicatricial pemphigoid and Stevens–Johnson syndrome. Am J Ophthalmol 122:38–52

    Google Scholar 

  39. Tsubota K, Goto E, Fujita H, Ono M, Inoue H, Saito I, Shimmura S (1999) Treatment of dry eye by autologous serum application in Sjogren’s syndrome. Br J Ophthalmol 83:390–395

    Google Scholar 

  40. Tsubota K, Goto E, Shimmura S, Shimazaki J (1999) Treatment of persistent corneal epithelial defect by autologous serum application. Ophthalmology 106:1984–1989

    Google Scholar 

  41. Tsubota K, Higuchi A (2000) Serum application for the treatment of ocular surface disorders. Int Ophthalmol Clin 40:113–122

    Google Scholar 

  42. Ubels JL, Iorfino A, O’Brien WJ (1991) Retinoic acid decreases the number of EGF receptors in corneal epithelium and Chang conjunctival cells. Exp Eye Res 52:763–765

    Google Scholar 

  43. Vesaluoma M, Teppo AM, Gronhagen-Riska C, Tervo T (1997) Release of TGF-beta 1 and VEGF in tears following photorefractive keratectomy. Curr Eye Res 16:19–25

    Google Scholar 

  44. Wilson SE, Chen L, Mohan RR, Liang Q, Liu J (1999) Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding. Exp Eye Res 68:377–397

    Google Scholar 

  45. Zimmermann R, Jakubietz R, Jakubietz M, Strasser E, Schlegel A, Wiltfang J, Eckstein R (2001) Different preparation methods to obtain platelet components as a source of growth factors for local application. Transfusion 41:1217–1224

    Google Scholar 

Download references

Acknowledgements

The authors thank Kaoru Araki-Sasaki, Tane Memorial Eye Hospital, Japan, and Fiona Stapleton, University of New South Wales, Australia, for providing HCE-T cell line; Gudrun Müller, Department of Ophthalmology, University of Lübeck, for technical assistance; Gudrun Knebel and Harry Manfeldt, Institute of Anatomy, University of Lübeck, for excellent support in SEM.

Author information

Authors and Affiliations

  1. Department of Ophthalmology, University of Lübeck, Ratzeburger Allee 160, 23538, Lübeck, Germany

    L. Liu, S. Harloff, P. Herminghaus & G. Geerling

  2. Institute of Immunology and Transfusion Medicine, University of Lübeck, Lübeck, Germany

    D. Hartwig

  3. Institute of Anatomy, University of Lübeck, Lübeck, Germany

    T. Wedel

Authors
  1. L. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. Hartwig
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. Harloff
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P. Herminghaus
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T. Wedel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. G. Geerling
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G. Geerling.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, L., Hartwig, D., Harloff, S. et al. An optimised protocol for the production of autologous serum eyedrops. Graefe's Arch Clin Exp Ophthalmol 243, 706–714 (2005). https://doi.org/10.1007/s00417-004-1106-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00417-004-1106-5

Keywords

Search

Navigation