Human platelet lysate produced from leukoreduction filter contents enables sufficient MSC growth

Abstract

Background Stem cell therapy holds significant potential for promoting recovery, with numerous products currently under development. Blood-derived supplements are often essential for successful stem cell expansion, with fetal bovine serum (FBS) being the most commonly used supplement. However, FBS has drawbacks, including the risk of immune responses, ethical concerns about animal welfare, and potential zoonotic infections. Human platelet lysate (hPL), derived from lysed platelets, contains various growth factors and has been proposed as an alternative to FBS. However, obtaining sufficient human platelets for clinical use remains challenging. Leukoreduction filters, used during blood transfusion manufacturing to remove leukocytes, also retain significant amounts of platelets and plasma. This study investigates the feasibility and efficacy of filter-derived hPL (f-hPL) for mesenchymal stem cell (MSC) expansion. Methods Leukoreduction filters were collected after their use in the manufacturing of whole blood transfusion products. Each filter was reverse-perfused with saline to extract residual blood contents. Platelets (f-platelet) and supernatant were separated by multiple centrifugation steps. f-Platelet were lysed with varying concentrations of fresh frozen plasma (FFP) to determine the optimal protein concentration for the lysate solution. Then, plasma left in the leukoreduction filters were used to generate lysate solution (f-plasma) at optimal protein concentration. f-Platelet (1.1 × 10^9/mL) and f-plasma (27 mg/mL protein) were combined in a freezing bag and subjected to three freeze-thaw cycles to produce f-hPL. Both small- and large-scale f-hPL were manufactured, and MSCs expansion and quality assessments were perfomed to evaluate the efficacy of f-hPL. Results A total of 3.5 ± 0.6 × 10^10 f-platelets were obtained from a single leukoreduction filter, yielding a collection efficiency of 37.1 ± 5.3%. The optimal protein concentration of lysate solution for cell expansion was > 27 mg/mL. Subsequently, six leukoreduction filters used to produce enough f-platelet and p-plasma for 100 mL of f-hPL. MSCs cultured in medium supplemented with 10% f-hPL demonstrated superior expansion, with cell proliferation rates 20% higher than those observed with commercial hPL and 300% higher than those cultured with FBS. The expanded MSCs met the International Society for Cell & Gene Therapy criteria for cell surface markers and differentiation potential.

1. Introduction

Cell transplantation therapy holds promise for promoting recovery in various diseases. To date, several cell types have been investigated, including embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells (MSC). In most cases, blood-derived supplements are required to be added in the culture medium to successfully expand stem cells, with fetal bovine serum (FBS) being the most commonly used supplement [1]. However, FBS poses potential risks, including zoonotic contamination, immunological reactions to xenogeneic serum antigens, and ethical concerns regarding animal welfare [2–4].

An alternative candidate is human AB serum, which offers the advantage of non-zoonotic contamination, and many clinical trials are currently utilizing human AB serum for cell expansion [5, 6]. However, due to its reliance on human resources, the cost of this supplement is relatively high, and its supply may be insufficient to meet the growing demand for industrial-scale stem cell products. Recent reports have demonstrated that human platelet lysate (hPL) is a promising substitute for cell supplement [7, 8], and several clinical trials have incorporated hPL as a supplement in stem cell culture media [9–15]. Similar to human AB serum, the preparation of a sufficient quantity of hPL is crucial for clinical applications. However, human platelets are primarily collected at blood banks for transfusion purposes, rather than for hPL production. Consequently, outdated platelet concentrates, which account for 2–4% of the total platelet concentrates, are frequently utilized for hPL production. Due to ongoing efforts to minimize outdated blood products, the availability of such platelet concentrates is expected to decrease, potentially limiting their use as a source for hPL.