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This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.
There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient’s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.
Regenerative medicine aims to heal or replace tissues and organs damaged by age, disease, or trauma as well as correct congenital defects. In autologous therapy, cells or tissue are withdrawn from a patient, expanded or modified, then reintroduced to the donor. This form of therapeutics has broad potential in the field of dermatology1. In autologous fibroblast therapy, a patient’s fibroblasts are cultured and reinjected to treat wrinkles, rhytids, or acne scars. As fibroblasts are the main functional cells in the dermis, injection of autologous fibroblasts may be more beneficial than other therapies in facial rejuvenation
The study protocol complied with the Declaration of Helsinki, and all patients provided written informed consent before participating in the study. Skin samples are obtained from healthy women undergoing abdominoplasty in the Plastic, Reconstructive and Aesthetic Surgery Department at Geneva University Hospitals (Geneva, Switzerland). The procedure conforms to the principles of the Declaration of Helsinki and was approved by the local institutional ethics committee (protocol #3126).
1. Preparati.......
This patented technology is a simple, fast, and reproducible medical device used to produce standardized PRP preparations. It is a one-step, fully closed system that allows the preparation of PRP from venous whole blood after 5 min of centrifugation at 1,500 x g (due to the separating gel technology). The PRP obtained after centrifugation is cleared from red and white blood cells, which sit below the gel. After several tube inversions, the platelets that are on top of the gel are resuspended in the plasma, and t.......
The advantages of using autologous fibroblasts as a natural alternative compared to other filler materials in wound cell therapy include good biocompatibility, minimal side effects, and easiness of harvesting and use. However, before using these therapeutics in a daily clinical setting, proper preclinical studies are necessary to identify the growth features and assess the biological function and safety of isolated fibroblasts both before and after transplantation. Thus, directly after the isolation process, in vitro exp.......
We thank Mr Grégory Schneiter for technical assistance with flow cytometry data; Professor Muriel Cuendet (Laboratory of Pharmacognosy, School of Pharmaceutical Sciences, and University of Geneva) for allowing the use of the Attune flow cytometer and the Cytation 3 high-throughput microscope; Professor Brigitte Pittet for scientific advices.
....Name | Company | Catalog Number | Comments |
96 well black clear flat bottom | BD Falcon | 353219 | 32/case |
Cell trace Violet Dye | Thermo Fischer Scientific | C34557 | 180 assays |
CuteCell PRP | Regen Lab SA | CC-PRP-3T | 3 tubes per package |
DAPI | Sigma | D9542 | 1 mg |
DMEM | Gibco | 52400-025 | 500 mL |
FBS | Gibco | 10270106 | 500 mL |
Glutamine 200 mM | Gibco | 25030024 | 100 mL |
Hematology Counter | Sysmex | KK-21N | |
Heparin 5000E Liquemine | Drossapharm AG | 0.5 mL | |
HEPES Buffer Solution 1M | Gibco | 15630-056 | 100 mL |
Liberase DH | Roche | 5401054001 | 2x 5 mg per package |
MEM NEAA 100x | Gibco | 11140-035 | 100 mL |
Na Pyruvate 1mg/mL | Gibco | 11360-039 | 100 mL |
Penicillin streptomycin | Gibco | 15140122 | 100 mL |
Phalloidin alexa Fluor 488 | Molecular Probes | A12379 | 300 units |
RPMI | Gibco | 31966-021 | 500 mL |
Trypsin 1x 0.25% | Gibco | 25050-014 | 100 mL |
Trypsin EDTA 0.25% | Gibco | 25200056 | 100 mL |
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