Human Platelet Lysate as Alternative of Fetal Bovine Serum for Enhanced Human In Vitro Bone Resorption and Remodeling

Abstract

IntroductionTo study human physiological and pathological bone remodeling while addressing the principle of replacement, reduction and refinement of animal experiments (3Rs), human in vitro bone remodeling models are being developed. Despite increasing safety-, scientific-, and ethical concerns, fetal bovine serum (FBS), a nutritional medium supplement, is still routinely used in these models. To comply with the 3Rs and to improve the reproducibility of such in vitro models, xenogeneic-free medium supplements should be investigated. Human platelet lysate (hPL) might be a good alternative as it has been shown to accelerate osteogenic differentiation of mesenchymal stromal cells (MSCs) and improve subsequent mineralization. However, for a human in vitro bone model, hPL should also be able to adequately support osteoclastic differentiation and subsequent bone resorption. In addition, optimizing co-culture medium conditions in mono-cultures might lead to unequal stimulation of co-cultured cells.MethodsWe compared supplementation with 10% FBS vs. 10%, 5%, and 2.5% hPL for osteoclast formation and resorption by human monocytes (MCs) in mono-culture and in co-culture with (osteogenically stimulated) human MSCs.Results and DiscussionSupplementation of hPL can lead to a less donor-dependent and more homogeneous osteoclastic differentiation of MCs when compared to supplementation with 10% FBS. In co-cultures, osteoclastic differentiation and resorption in the 10% FBS group was almost completely inhibited by MSCs, while the supplementation with hPL still allowed for resorption, mostly at low concentrations. The addition of hPL to osteogenically stimulated MSC mono- and MC-MSC co-cultures resulted in osteogenic differentiation and bone-like matrix formation, mostly at high concentrations.ConclusionWe conclude that hPL could support both osteoclastic differentiation of human MCs and osteogenic differentiation of human MSCs in mono- and in co-culture, and that this can be balanced by the hPL concentration. Thus, the use of hPL could limit the need for FBS, which is currently commonly accepted for in vitro bone remodeling models.

Evaluating material-driven regeneration in a tissue engineered human in vitro bone defect model

“…To replace FBS, alternatives for HUVEC-hBMSC co-cultures like human platelet lysate or defined serum-free media need to be explored. Human platelet lysate was already demonstrated to support bone remodeling [66], while serum-free medium proved efficient for HUVECs co-cultured with human adipose tissue derived stromal cells [67].…”

A dialysis medium refreshment cell culture set-up for an osteoblast-osteoclast coculture

“…Briefly, SF scaffolds were incubated with a cell suspension (3×10 5 cells/4 mL expansion medium) in 50-mL tubes placed on an orbital shaker at 150 rpm for 6 hours in an incubator at 37°C and 5% CO 2 . Next, the samples were transferred to the dialysis wells plates and incubated at 37°C and 5% CO 2 for a total 4 weeks in osteogenic monoculture medium containing DMEM (low glucose, 22320, Thermo Scientific), 10% human platelet lysate [11] (hPL, PE20612, PL BioScience, Aachen, Germany), 1% Anti-Anti, 0.1 μM dexamethasone (D4902, Sigma-Aldrich), 0.05 mM ascorbic acid-2-phosphate (A8960, Sigma-Aldrich), 10 mM β-glycerophosphate (G9422, Sigma-Aldrich). The insert of the dialysis wells plates contained the scaffold and 200 μL osteogenic medium, the base contained 500 μL osteogenic medium.…”

“…Moreover, a 2-10 month culture period would generate practical issues for high throughput drug testing. Therefore, we propose a fully human 3D bone-remodeling model, including the use of human platelet lysate (hPL) instead of the generally used fetal bovine serum (FBS) [11]. We present a simple, cost-effective method for incorporating dialysis into the cell culture.…”

The impact of culture variables on 3D humanin vitrobone remodeling; a design of experiments approach

“…For mechanical loading, static and dynamic loading were included, using a custom made spinner flask bioreactor at 300 RPM to apply fluid shear stress [21]. As a serum supplement, hPL was used at 5% and 10% concentration since the most commonly used FBS can inhibit osteoclast resorption [11,12]. For osteogenic supplements, ascorbic acid was used in all conditions as a requirement for collagen synthesis.…”

“…Researchers have already attempted to study the influences of culture variables on human osteoblast-osteoclast co-cultures. For example, studies looked at the influence of cell ratio on osteoclast formation [8], osteogenic factor addition and timing on osteogenic and osteoclastic differentiation [9,10], and the replacement of the culture supplement fetal bovine serum (FBS) by serum free medium [11], or human platelet lysate (hPL) [12] on osteoclastic resorption. As such, most studies analyze the influence of only one culture variable on in vitro remodeling outcomes while a specific combination of multiple variables might lead to improved results.…”