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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This article describes a protocol to simplify the process and render the preparation of autologous conditioned serum (ACS) less expensive. No special syringes or surface-coated glass beads are needed. Moreover, the modified ACS (mACS) has competitive advantages over conventional autologous serum in the corneal wound healing of murine eyes ex vivo.

Abstract

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched in epitheliotropic growth factors that are essential in corneal wound healing. Unlike AS, PRP is based on a differential centrifugation system, yielding more platelet-derived growth factors. Autologous conditioned serum (ACS) not only preserves the preparation of AS and PRP, but also focuses on immune-modulating properties, which are important in inflammatory diseases.

The lack of standardized protocols and high preparation costs are limitations for the clinical application of ACS. This video experiment demonstrates a standard operating procedure for preparing modified autologous conditioned serum (mACS) eye drops. First, glycerol was added into heparin syringes as the blood cell stabilizer during hypoxic incubation. To activate the blood cells, a 4 h incubation at 37 °C was initiated. Then, the blood samples were centrifuged at 3,500 × g for 10 min at room temperature. After filtration of the supernatant through a 0.22 µm filter, the mACS eye drops were fully prepared.

A tentative try-out of the therapeutic effect of mACS showed that it may have competitive advantages over conventional AS in the corneal wound healing in ex vivo mouse eyes. The AS used in this study was prepared according to published studies and the clinical practice in our hospital. Therefore, the efficacy of mACS on ocular surface diseases could be evaluated in future research through in vivo animal studies and clinical trials.

Introduction

The therapeutic effects of autologous serum (AS) in dry eye diseases were first reported in the 1980s by Fox et al.1. It is believed that both the lubricating property and the essential epitheliotropic biochemical components in AS, mimicking natural tears, benefit the proliferation of corneal epithelial cells. Over the past decades, several studies have been performed on this basis. Trophic components include epidermal growth factor (EGF), vitamin A, transforming growth factor β (TGF- β), and other cytokines. Interestingly, the serum is rich in TGF- β and vitamin A, which are believed to play a pivotal role in epidermal prolifera....

Protocol

The research was performed in compliance with institutional guidelines at the beginning of the protocol section. All protocols and procedures were carried out according to the Declaration of Helsinki and were reviewed and approved by the Chang Gung Medical Foundation Institutional Review Board. All volunteers were informed of the nature of this study and signed an informed consent form prior to their inclusion. The consumables required for the entire experimental procedure are presented in Figure 1

Representative Results

Figure 1 and Figure 2 show the materials needed for the experiment, and Figure 3 displays the sequential steps and the successful mid-products during the preparation of mACS. First, 0.5 mL of 10% glycerol solution was added into each 10 mL sterile test tube (Figure 3A). Then, 60-70 mL of venous blood was obtained from the patient, and 10 mL of blood was injected into each tube (

Discussion

In this study, a protocol for the preparation of mACS is described and the benefit of mACS eye drops in the wound healing of animal models is further shown. The crucial modification of this mACS protocol is the addition of approximately 0.5 mL of 10% glycerol solution in each test tube, which creates suitable hypoxic conditions during the 4 h incubation at 37 °C. This setting provides the AS with proper stress and prompt cells to secrete the necessary growth factors that help wound healing. The 0.22 µm filter c.......

Acknowledgements

The authors thank Ya-Lan Chien and Chia-Ying Lee for excellent technical assistance, and OnLine English company for the linguistic edition. This study was funded in part by Chang Gung Medical Research Project (Grant No. CMRPG3L1491).

....

Materials

NameCompanyCatalog NumberComments
 96-well culture plateMerck KGaA, GermanyCLS3997
Barraquer lid speculumkatenaK1-535515 mm
Barraquer needle holderKatenaK6-3310without lock 
Barron Vacuum Punch 8.0 mmkatenaK20-2108for cutting filter paper
BD 10.0 mL vacutainer tubes containing heparin 158 USP unitsBecton,Dickinson and Company, US367880At least 6 tubes, necessary to collect blood for subsequent experiments and to avoid blood agglutination
BD 21 G butterfly-winged infusion setBecton,Dickinson and Company, US367281For even distribution of glycerol solution
C57BL/6 mice National Laboratory Animal CenterRMRC11005for mouse model
Castroviejo forceps 0.12 mmkatena K5-2500
CentrifugeEppendorf, Germany58110004283,500 x g for 10 min
Cheng Yi 10.0 mL sterilized eye dropper bottleCheng Yi Chemical, TaiwanCP405141Must be sterile and as the storage container for the final product
Corneal rust ring remover with 0.5 mm burrAlgerbrush IITM; Alger Equipment Co., Inc. Lago Vista, TXCHI-675for debridement of the corneal epithelium
Dulbecco's modified minimal essential mediumMerck KGaA, GermanyD6429
Filter paper Toyo Roshi Kaisha,Ltd.1.11
Fluorescein sodium ophthalmic strips U.S.POPTITECHOPTFL100staining for corneal epithelial defect 
IncubatorFirstek, TaiwanS300S37 °C for 4 h
Kanam sterile glovesKanam Latex Industries, IndiaEN455For aseptic operation
Merck 0.22 µm filterMerck KGaA, GermanyPR05359At least 2 filters for mACS filtration
Nang Kuang 250 mL 10% glycerol solutionNang Kuang Pharmaceutical, Taiwan19496To offer suitable membrane stabilization effect and extracellular osmotic pressure for blood cells
Normal salineTAIWAN BIOTECH CO., LTD.100-120-1101
Skin biopsy punch 2mmSTIEFEL22650
StereomicroscopeCarl Zeiss Meditec, Dublin, CASV11microscope for surgery
Terumo 18 G needleTerumo, TaiwanSMACF0120-18BX3.0 mL syringe with 18 G needle to extract the supernatant after centrifugation
Terumo 20.0 mL syringeTerumo, TaiwanMDSS20ESCould be used to collect serum after initial centrifugation and use it for secondary centrifugation.
Terumo 3.0 mL syringe with the 23 G needleTerumo, TaiwanMDSS03S23253.0 mL syringe is used to extract the supernatant after centrifugation. Then connect the filter and the 23 G needle for injection into the eye drop bottles.
Westcott Tenotomy Scissors MediumkatenaK4-3004

References

  1. Fox, R. I., Chan, R., Michelson, J. B., Belmont, J. B., Michelson, P. E. Beneficial effect of artificial tears made with autologous serum in patients with keratoconjunctivitis sicca. Arthritis and Rheumatology. 27 (4), 459-461 (1984).
  2. Noble, B. A., et al.

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