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* These authors contributed equally
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.
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.
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....
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
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 (
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.......
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).
....Name | Company | Catalog Number | Comments |
96-well culture plate | Merck KGaA, Germany | CLS3997 | |
Barraquer lid speculum | katena | K1-5355 | 15 mm |
Barraquer needle holder | Katena | K6-3310 | without lock |
Barron Vacuum Punch 8.0 mm | katena | K20-2108 | for cutting filter paper |
BD 10.0 mL vacutainer tubes containing heparin 158 USP units | Becton,Dickinson and Company, US | 367880 | At least 6 tubes, necessary to collect blood for subsequent experiments and to avoid blood agglutination |
BD 21 G butterfly-winged infusion set | Becton,Dickinson and Company, US | 367281 | For even distribution of glycerol solution |
C57BL/6 mice | National Laboratory Animal Center | RMRC11005 | for mouse model |
Castroviejo forceps 0.12 mm | katena | K5-2500 | |
Centrifuge | Eppendorf, Germany | 5811000428 | 3,500 x g for 10 min |
Cheng Yi 10.0 mL sterilized eye dropper bottle | Cheng Yi Chemical, Taiwan | CP405141 | Must be sterile and as the storage container for the final product |
Corneal rust ring remover with 0.5 mm burr | Algerbrush IITM; Alger Equipment Co., Inc. Lago Vista, TX | CHI-675 | for debridement of the corneal epithelium |
Dulbecco's modified minimal essential medium | Merck KGaA, Germany | D6429 | |
Filter paper | Toyo Roshi Kaisha,Ltd. | 1.11 | |
Fluorescein sodium ophthalmic strips U.S.P | OPTITECH | OPTFL100 | staining for corneal epithelial defect |
Incubator | Firstek, Taiwan | S300S | 37 °C for 4 h |
Kanam sterile gloves | Kanam Latex Industries, India | EN455 | For aseptic operation |
Merck 0.22 µm filter | Merck KGaA, Germany | PR05359 | At least 2 filters for mACS filtration |
Nang Kuang 250 mL 10% glycerol solution | Nang Kuang Pharmaceutical, Taiwan | 19496 | To offer suitable membrane stabilization effect and extracellular osmotic pressure for blood cells |
Normal saline | TAIWAN BIOTECH CO., LTD. | 100-120-1101 | |
Skin biopsy punch 2mm | STIEFEL | 22650 | |
Stereomicroscope | Carl Zeiss Meditec, Dublin, CA | SV11 | microscope for surgery |
Terumo 18 G needle | Terumo, Taiwan | SMACF0120-18BX | 3.0 mL syringe with 18 G needle to extract the supernatant after centrifugation |
Terumo 20.0 mL syringe | Terumo, Taiwan | MDSS20ES | Could be used to collect serum after initial centrifugation and use it for secondary centrifugation. |
Terumo 3.0 mL syringe with the 23 G needle | Terumo, Taiwan | MDSS03S2325 | 3.0 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 Medium | katena | K4-3004 |
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