We thank Mr Gr&#233;gory 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.

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. Preparation of PRP
NOTE: The CuteCell-PRP tubes (Table of Materials) are designed for the rapid preparation of PRP from a small volume of the patient&#8217;s blood in a closed circuit system.
<ol>
	<li>Collection of whole blood 
	NOTE: Harvest autologous blood from a peripheral vein of the arm using a butterfly needle directly connected to the PRP tube, according to the collection protocol of the medical institute. Blood can be directly withdrawn through the venous cannula if the patient is under anesthesia.
	<ol>
		<li>Open the tube blister pack. Perform the venous puncture and fill the desired number of PRP tubes with whole blood. The vacuum within the tubes will enable automatic collection of the necessary volume of blood (~10 mL).</li>
		<li>Carefully turn the tubes upside down several times to mix the blood with anticoagulant.</li>
		<li>Inside a biosafety cabinet (class 2), remove 100 &#181;L of total whole blood using a 1 mL syringe for further blood cell number counts.</li>
	</ol>
	</li>
	<li>Centrifugation 
	NOTE: Ensure that the centrifuge is correctly balanced before starting it.
	<ol>
		<li>Once the blood is collected in the PRP tubes, if necessary, prepare a counterbalance tube (supplied separately) with water to the same level as the blood in the PRP tube. Place the filled tubes in the centrifuge opposite each other, ensuring that the machine is balanced.</li>
		<li>Centrifuge samples at 1,500 x g for 5 min. 
		NOTE: Set the corresponding rpm speed according to the centrifuge manufacturer&#8217;s instructions. After centrifugation, the blood will become fractionated. The red and white blood cells are trapped under the gel, and platelets settle on the surface of the gel (Figure 1).</li>
	</ol>
	</li>
	<li>Gently invert the PRP tube 20x to resuspend the platelet deposit in the plasma supernatant. 
	NOTE: Ensure that the platelets are fully detached from the gel. The plasma should change from clear and transparent to turbid. If platelet aggregates are present, they should be collected with the plasma.</li>
	<li>To collect PRP, take the plasma solution from the tube using a syringe transfer device connected to a 10 mL syringe. 
	NOTE: About 5 mL of PRP will be obtained from each tube. The PRP solution is now ready to mix in the final medium preparation. The solution can be kept at room temperature (RT) under the safety cabinet until the future steps involving hematology analyzer and use.</li>
	<li>Before using the PRP, determine the platelet concentration, mean platelet volume, and number of red and white blood cells using an automated hematology analyzer (Table of material). To do so, carefully withdraw 100 &#181;L of the solution and transfer into a 1.5 mL tube. 
	NOTE: PRP is used as an autologous culture supplement at a concentration between 5&#8211;20% v/v. The optimal concentration of PRP needs to be determined for each cell line. To prevent fibrin clot formation, the final culture media should be supplemented with 2 U/mL heparin.</li>
</ol>
2. Isolation of autologous fibroblasts and culture in FBS- or PRP-supplemented media
<ol>
	<li>Wash skin samples in phosphate-buffered saline (PBS) by gently shaking in a 50 mL polypropylene tube.</li>
	<li>If the skin sample is relatively large (&#62;10 cm2), place the sample on the lid of a 150 cm2 tissue culture dish (epidermal side down). Remove the subcutaneous fat tissue using a pair of forceps and scissors. To prevent the tissue from drying out, rinse the tissue every few minutes in PBS. 
	NOTE: Use smaller tissue culture dish for smaller samples.</li>
	<li>When fat trimming is complete, cut the tissue into approximately 0.5 cm x 1.5 cm strips using a sterile scalpel.</li>
	<li>Add 5 mL of collagenase-dispase mix (Table of Materials; 14 W&#252;nsch units/mL) to a sterile 15 mL tube.</li>
	<li>Transfer the cut tissue to the tube, ensuring that the tissue pieces are submerged in the solution. Cap the tube securely.</li>
	<li>Place the tubes in an incubator at 37 &#176;C with orbital shaking for 150 min. After incubation, place the tube containing the tissue in the biosafety cabinet.</li>
	<li>Place the lid of a sterile 100 mm culture dish upside down in the biosafety cabinet. Transfer the digested tissue solution to the bottom of the 100 mm culture dish, without splashing. If any pieces of tissue remain in the tube, use a sterile 1 mL pipette or sterile forceps to transfer the tissue pieces to the bottom of the 100 mm culture dish.</li>
	<li>With the epidermis strip facing up, separate the intact epidermis sheet from the dermis by using two pairs of forceps. Hold the dermis of the tissue strip with a pair of forceps and the edge of the epidermis with another pair of forceps. Peel the dermis and epidermis apart in two pieces, keeping the separated pieces on the same lid. Try to perform this step quickly and repeat the manipulation for each piece of tissue.</li>
	<li>Transfer the dermal pieces to a new 100 mm culture dish containing PBS.</li>
	<li>Using laboratory forceps, place the dermal pieces in a 15 mL polypropylene tube with 3 mL of 0.3% trypsin/PBS. Incubate for 10-20 min in a 37 &#176;C water bath and invert the tube several times every 2&#8722;3 min.</li>
	<li>To stop the enzymatic reaction, add 3&#8722;5 mL of ice-cold complete growth medium (Dulbecco&#8217;s modified Eagle medium [DMEM] or RPMI containing 10% FBS). Vortex the tube vigorously several times. Filter the fibroblast suspension through an 85 &#956;m nylon mesh (placed over the top of a 50 mL tube) to remove dermal debris.</li>
	<li>Centrifuge for 10 min at 150 x g, at 4 &#176;C. Aspirate the supernatant and resuspend the pellet in 100&#8722;1,000 &#956;L of complete growth medium.</li>
	<li>Using trypan blue (dilution factor of x2) mixed with the cell suspension, count the number of total and viable cells by filling the chamber of the haemocytometer. 
	NOTE: Cell viability depends on the conditions used for enzymatic digestion. To increase cell recovery and cell viability, the skin sample may be cut into smaller pieces.</li>
	<li>Plate 3&#8722;10 x 104 cells in 5 mL of complete FBS growth medium (DMEM, 10% FBS heat-inactivated for 60 min at 56 &#176;C, 1% 1 M HEPES buffer solution, 1% 100x nonessential amino acid mixture, 1% 100x L-glutamine, 1% 100x penicillin/streptomycin, 1% 100x sodium pyruvate) in a 25 cm2 tissue culture flask. Incubate at 37 &#176;C. 
	NOTE: Viable fibroblasts will attach to the flask within 24 h and begin to exhibit the spindle-shape in 2&#8722;3 days.</li>
	<li>On day 2, aspirate the medium containing nonadherent cells and add fresh medium. 
	NOTE: As dead cells in the culture medium affect the growth of viable fibroblasts, nonadherent, dead cells must be removed from the culture.</li>
	<li>Change the medium every 3&#8722;4 days until the culture reaches 70&#8722;80% confluency.</li>
	<li>To harvest fibroblasts, wash with PBS and incubate with trypsin/EDTA solution for 3 min at 37 &#176;C in the incubator.</li>
	<li>To stop the reaction, add 3&#8722;5 mL of warm complete growth medium (DMEM or RPMI containing 10% FBS). Take an aliquot of the cell suspension to count the cells with the haemocytometer. Centrifuge the suspension for 5 min at 200 x g and remove the medium by aspiration.</li>
	<li>Culture the fibroblast in PRP medium (DMEM, 20% PRP, 1% 1 M HEPES buffer solution, 1% 100x nonessential amino acid mixture, 1% 100x L-glutamine, 1% 100x penicillin/streptomycin, 1% 100x sodium pyruvate, 2 U/mL heparin) in the incubator at 37 &#176;C. 
	NOTE: The minimum cell density recommended is 4,000 viable cells/cm2.</li>
	<li>To assess PRP effects on fibroblast proliferation, seed fibroblasts (passage 2) in 24 well plates at a density of 8 x 103 cells per well in PRP medium with different PRP concentrations (1%, 5%, 10%, 20%, 30%, 40%, and 50%) and with 2 U/mL heparin or under classical culture medium conditions (10% FBS). After 7 days of culture, add a vital dye (cell proliferation violet) to cells and assess the proliferation by flow cytometry.</li>
	<li>To study cytoskeletal rearrangements, seed cells in 96 well black/clear flat bottom plates at a concentration of 1 x 105 cells/mL in complete FBS growth DMEM (see step 2.14) supplemented with 0.5% FBS for 24 h. Treat cells with 10% FBS or different PRP concentrations (1%, 5%, 10%, 20%, 30%, 40%, and 50%) for 7 days.&#160;
	<ol>
		<li>Fix the fibroblasts with 4% paraformaldehyde for 10 min and permeabilize with 0.1% Triton X-100 for 5 min at RT. Stain the fibroblasts with 50 mL of 5 U/mL phalloidin, wash 2x with PBS, and mark with 50 mL of 1 mg/mL 4&#8242;,6-diamidino-2-phenylindole (DAPI) for 5 min.&#160;Cytation 3 cell imaging multimode reader (BioTek) was used to visualize phalloidin staining.</li>
	</ol>
	</li>
</ol>

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This project has been funded by Regen Lab SA. Sarah Berndt is the cell therapy head project manager for Regen Lab and is employed by Regen Lab SA. Antoine Turzi is the CEO of RegenLab.

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 expansion of the cells must be performed quickly in order to limit the number of cell divisions. Culture conditions must be tightly controlled to ensure the safety and efficacy of the final autologous biological product.
In published preclinical studies, cells at passages 3&#8722;4 have typically been chosen to be injected, as they are most suitable in terms of cell quantity and proliferative/secretory activity15. Most preclinical research has been performed with culture media in which FBS is the source of nutrients and growth factors16. However, this xenogeneic growth supplement presents a possible risk of infection and immunological reactions. Due to the occurrence of bovine spongiform encephalopathy (BSE) in herds worldwide, there is considerable regulatory concern regarding the use of FBS as a cell culture supplement because of the risk of introducing xenogeneic material into cell therapy cultures10.
Usually, proliferation of fibroblast or mesenchymal stem cells (MSC) are achieved using FBS- supplemented growth media. While FBS has been an adequate nutrient supply for cell culture, it poses additional hindrances besides the risk of zoonose contamination. First, FBS products present huge manufacturing cost compared to human blood-derived products, partly due to reduced proliferation rates and an extended culture expansion period9. Second, FBS is a source of xenoproteins that interact with the isolated cells, thereby increasing the risk of immune reaction because of the appearance of antibodies against surface protein complexes on transplanted cells10.
To address the potential drawbacks of FBS as a nutrient for clinical expansion of cells, human blood-derived products have been introduced as alternatives. Some studies have proposed replacing FBS in culture media with autologous human serum for cell expansion, but since cell proliferation is slow, it still requires a long culture time (3 weeks) and several cell passaging before transplantation17,18.
Other groups have used platelet lysate (PL), a cell-free supernatant rich in growth factors that are released from platelets after freeze-thawing disruption of the platelet concentrates. The main advantage of PL compared to PRP is its frozen storage potential. Therefore, the same lot can be used for consecutive cell culture applications. Furthermore, as an off-the-shelf product, it can be standardized by analyzing the growth factor content and other biological features before use. Some disadvantages are that it is allogenic, and batches are pooled from several donors. Moreover, since PL does not contain any living platelets, which can produce cytokines and growth factors for up to 10 days, culture media containing PL has to be changed every 3 days (instead of 7&#8722;10 days when culture is supplemented with PRP)19. Expanding the cells in vitro in their own source of growth factors and nutrients provided by this PRP preparation is certainly advantageous. The preparation is free of any xenogeneic supplements in the cell culture that could cause rejection, allergy, or transmission of infectious disease.
In previous studies, it has been demonstrated that autologous PRP can be used as a safe, efficient, and cost-effective culture medium for adipose-derived mesenchymal stem cell (ADSC) proliferation20. Media supplied with 20% autologous PRP increased cell proliferation up to 13-fold without changing the cell phenotype. In clinics, PRP is considered to have powerful rejuvenation and regeneration capacities.
Currently, PRP obtained from a patient&#8217;s blood is already used efficiently in the clinical setting for wound healing alone21 or in combination with hyaluronic acid22,23 or bone regeneration24,25; or skin rejuvenation alone26 or in combination with adipose-derived stromal vascular fraction cells27,28,29. This prompted the investigation of using a patient&#8217;s own PRP to promote expansion of autologous fibroblasts.
For this purpose, and to ensure reproducibility of the results obtained with the PRP preparation, we developed a special PRP device. This closed system allows the standardization of the PRP preparations, which is a prerequisite to minimize fluctuations from a patient&#8217;s donor (regarding platelet concentration and operator-dependent variations). The blood cell counts obtained from 10 patients showed that the protocol is highly reproducible (Figure 2). Moreover, it was shown that PRP had potent effects as a proliferation booster, up to 7.7-fold compared to FBS (Figure 3B). This is important in order to reduce the costs and time required for the expansion process. In a recent study, we further analyzed how PRP affects fibroblast proliferation and differentiation. The 20% PRP displayed the best efficacy without affecting the cell phenotype or genotype14.
A limitation of the method is that the medium containing PRP must be prepared with heparin to prevent clot formation. Even though standard plasma anticoagulant (a sodium citrate anticoagulant and calcium chelator) is used in this device, fibrin clots formation can occur when plasma is added to culture media that contains calcium30. Heparin addition to the PRP preparation prevents this fibrin clot formation. However, heparins can bind growth factors and may interfere with cell growth30. For example, heparin was shown to negatively affect dermal fibroblast proliferation31. However, the concentrations used were 4x more concentrated compared to the concentration used in this study (2 U/mL).
Moreover, commercial heparin is mainly purified from porcine sources; therefore, because of its animal origin, it represents a limitation in the development of completely xeno-free medium. Although porcine heparin has been approved for human therapy, hypersensitivity to the molecule has been reported32. Another limitation is that PRP must be used as a fresh biological supplement and cannot be frozen. Concerning the half-life of the platelets, it has been previously shown that after 10 days, half of the platelets present in the PRP are still viable33. For this reason, the PRP is kept at room temperature and used for 10 days. Moreover, for future cell therapies that require the production of massive numbers of cells, the main limitation is the total amount of blood that can be withdrawn from the patient.
In summary, this protocol demonstrates that replacing FBS with PRP results in a rapid expansion of fibroblasts. The platelets present in the PRP slowly release an orchestrated mix of growth factors during the culture phase of expansion. The presented device is used during a standardized procedure that reduces the complexity and hands-on time during the preparation of PRP. This system is thus deemed an appropriate method when a quick and safe ex vivo expansion is needed for autologous cell therapy applications in GMP conditions.

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&#8217;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 rejuvenation2.
In the skin, fibroblasts are responsible for the synthesis and secretion of extracellular proteins (i.e., collagen, elastin, hyaluronic acid, and glycosaminoglycans). They also release growth factors that regulate cell function, migration, and cell-matrix/cell-cell interactions in normal skin homeostasis and wound healing3. Dermal fibroblasts have already been introduced as a potential clinical cell therapy for skin wound healing4, tissue regeneration5, or dermal fillers in esthetic and plastic surgery procedures6. Some studies even suggest that, in the context of regenerative medicine, fibroblasts may be a more practical and effective cell therapy than mesenchymal stem cells7.
To obtain a sufficient number of fibroblasts for clinical applications, cell expansion is usually mandatory. Ex vivo/in vitro cell culture requires basal medium supplemented with growth factors, proteins, and enzymes to support cell adhesion and proliferation. Fetal bovine serum (FBS) is a common supplement for cell culture media, because fetal blood 1) is rich in growth factors compared to adult blood and 2) presents a low antibody content8. As cell therapy progresses, there are concerns about the safety of classical cell culture conditions in which FBS is added to the culture medium. Furthermore, there is now a tendency to replace FBS with alternatives9. Several FBS substitutes have shown promising results10.
The platelet-rich plasma (PRP) alternative has been selected here, and we have developed a medical device to produce a standardized preparation of PRP, named CuteCell-PRP. The intended use of this device is the preparation of autologous PRP to be used as a culture media supplement for in vitro expansion of autologous cells under GMP conditions.
PRP is defined as a concentrated platelet suspension in plasma. Because there are numerous preparation protocols, which differ in 1) the amount of blood needed, 2) types of devices used, and 3) centrifugation protocol, the resulting platelet concentrations vary from slightly above to more than 10x the blood baseline value. In addition, PRP preparations contain variable levels of red and white blood cell contamination. The terminology &#8220;PRP&#8221; is thus used to describe products that vary greatly in their biological composition and potential therapeutic effects.
In most studies, FBS substitution is achieved using different concentrations of PRP that is activated (by thrombin or calcium). This artificial activation provokes an immediate and important release of platelet growth factors from 15 min up to 24 h11. Therefore, it is believed that platelet activation is undesirable for applications in cell cultures, in which the slow release of growth factors from gradual platelet degranulation is required.
PRP therapy involves the preparation of autologous platelets in concentrated plasma12. The optimal platelet concentration is unclear, and a broad range of commercial devices are available to prepare PRP13. This lack of standardization results from inconsistency among studies and has led to a black box regarding the dosage and timing of injection. This protocol describes a procedure to obtain autologous PRP using this dedicated PRP device to expand skin fibroblasts in a 100% autologous ex vivo culture model.

<table><tbody><tr><td>96 well black clear flat bottom</td><td>BD Falcon</td><td>353219</td><td>32/case</td></tr><tr><td>Cell trace Violet Dye</td><td>Thermo Fischer Scientific</td><td>C34557</td><td>180 assays</td></tr><tr><td>CuteCell PRP</td><td>Regen Lab SA</td><td>CC-PRP-3T</td><td>3 tubes per package</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>1 mg</td></tr><tr><td>DMEM</td><td>Gibco</td><td>52400-025</td><td>500 mL</td></tr><tr><td>FBS</td><td>Gibco</td><td>10270106</td><td>500 mL</td></tr><tr><td>Glutamine 200 mM</td><td>Gibco</td><td>25030024</td><td>100 mL</td></tr><tr><td>Hematology Counter</td><td>Sysmex</td><td>KK-21N</td><td></td></tr><tr><td>Heparin 5000E Liquemine</td><td>Drossapharm AG</td><td></td><td>0.5 mL</td></tr><tr><td>HEPES Buffer Solution 1M</td><td>Gibco</td><td>15630-056</td><td>100 mL</td></tr><tr><td>Liberase DH</td><td>Roche</td><td>5401054001</td><td>2x 5 mg per package</td></tr><tr><td>MEM NEAA 100x</td><td>Gibco</td><td>11140-035</td><td>100 mL</td></tr><tr><td>Na Pyruvate 1mg/mL</td><td>Gibco</td><td>11360-039</td><td>100 mL</td></tr><tr><td>Penicillin streptomycin</td><td>Gibco</td><td>15140122</td><td>100 mL</td></tr><tr><td>Phalloidin alexa Fluor 488</td><td>Molecular Probes</td><td>A12379</td><td>300 units</td></tr><tr><td>RPMI</td><td>Gibco</td><td>31966-021</td><td>500 mL</td></tr><tr><td>Trypsin 1x 0.25%</td><td>Gibco</td><td>25050-014</td><td>100 mL</td></tr><tr><td>Trypsin EDTA 0.25%</td><td>Gibco</td><td>25200056</td><td>100 mL</td></tr></tbody></table>

production of autologous platelet-rich plasma for boosting in vitro human fibroblast expansion

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&#8217;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.

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 the PRP is ready to use (Figure 1).
To assess the reproducibility of the blood cell content in the preparation, whole blood and PRP samples were analyzed from 10 different patients with a hematology analyzer. After processing of the whole blood with the device, the majority of the red blood cells (RBCs) and white blood cells (WBCs) were removed. The mean platelet concentration showed a 1.5-fold increase when compared to the physiological concentration found in the whole blood (Figure 2).
In the autologous culture set-up, a patient&#8217;s isolated fibroblasts were cultivated in the presence of increasing PRP concentrations (1&#8722;50%), compared to the classical culture medium condition (10% FBS). After 7 days of culture without changing the media, the cultures primed with 20% PRP exhibited a higher number of viable fibroblasts (Figure 3A,B; 7.7x higher compared to the control condition). Phalloidin staining evidenced that the morphological change observed upon-PRP activation was related to F-actin reorganization, which is a signature of fibroblast activation (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60816/60816fig01.jpg" /> 
Figure 1: Preparation of autologous PRP to be used as a culture media supplement for in vitro expansion of autologous cells under GMP conditions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60816/60816fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60816/60816fig02.jpg" /> 
Figure 2: Blood cell and platelet counts.&#160;PRP prepared with the commercial device displays highly reproducible blood cell content despite inter-donor variations. PLT: platelets (x 105); WBCs: white blood cells (x 103); RBCs: red blood cells (x 106), n = 10 patients. This figure has been modified from Berndt et al.14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60816/60816fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60816/60816fig03.jpg" /> 
Figure 3: Assessment of PRP proliferative effects on autologous fibroblast culture.&#160;(A) Representative brightfield microscopic images demonstrating the boosting effects of 20% PRP medium added to fibroblasts isolated from the same patient after 7 days of culture. Scale bar = 100 &#181;m. (B) Assessment of PRP proliferative effect by flow cytometry using vital dye. Proliferative effects of increasing PRP concentrations in comparison (1&#8722;50%) with 10% FBS (n = 10 patients) on normal human dermal fibroblast (NHDF) for 7 days without medium change in a complete autologous system (cells and PRP from the same patient; *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001). This figure has been modified from Berndt et al.14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60816/60816fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60816/60816fig04.jpg" /> 
Figure 4: Cytoskeleton visualization through F-actin staining.&#160;After 7 days grown in 20% PRP (right panel), fibroblasts are activated and reorganize their cytoskeleton as compared to control culture conditions with 10% FBS (left panel). Scale bar = 200 &#181;m. This figure has been modified from Berndt et al.14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60816/60816fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion at JoVE.com

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

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 ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

2.4K Views.  Geneva University. This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

Video: Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Immunology and Infection

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Developmental Biology

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Biology

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of cultured cells, implying a possible use in regenerative medicine. Here, a double-spin method was used to prepare PRP from whole blood. PRP was further activated by autologous thrombin. The platelet count was measured in the activated PRP and the proliferation-stimulating effect in human adipose-derived stem cells (hASCs) was examined. The resulting platelet count was 11.5-times higher in PRP than in whole blood plasma. The proliferation of hASCs was markedly enhanced by incubation with 1% PRP. The described method can be used to reproducibly prepare PRP with a high concentration of platelets. PRP prepared by this method markedly promotes proliferation of hASCs.

In this report, we describe a double-spin method for preparing activated platelet-rich plasma (PRP). Using autologous thrombin, human adipose-derived stem cells (hASCs) were cultured. Activated PRP was shown to promote proliferation of hASCs.

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

An In Vitro Method for the Differentiation of Megakaryocytes and Platelet Formation

Source: Perdomo, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56420">Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells.</a> J. Vis. Exp. (2017)
This video demonstrates a technique for the differentiation of megakaryocytes and the formation of platelets. Hematopoietic stem progenitor cells &#8212; isolated from human umbilical cord blood &#8212; differentiate into mature megakaryocytes in the presence of thrombopoietin. The mature megakaryocytes produce cytoplasmic extensions termed proplatelets, which give rise to individual platelets.

Source: Perdomo, J., et al. Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells. J. Vis. Exp. (2017)
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Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

Summary

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Chapters in this video

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34⁺ Cells

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

An In Vitro Method for the Differentiation of Megakaryocytes and Platelet Formation