Separation of in‐vitro‐derived megakaryocytes and platelets using spinning‐membrane filtration

Abstract

In-vitro-derived platelets (PLTs) could potentially overcome problems associated with donated PLTs, including contamination and alloimmunization. Although several groups have produced functional PLTs from stem cells in vitro, the challenge of developing this technology to yield transfusable PLT units has yet to be addressed. The asynchronous nature of in vitro PLT generation makes a single harvest point infeasible for collecting PLTs as soon as they are formed. The current standard of performing manual centrifugations to separate PLTs from nucleated cells at multiple points during culture is labor-intensive, imprecise, and difficult to standardize in accordance with current Good Manufacturing Practices (cGMP). In an effort to develop a more effective method, we adapted a commercially-available, spinning-membrane filtration device to separate in-vitro-derived PLTs from nucleated cells and recover immature megakaryocytes (MKs), the precursor cells to PLTs, for continued culture. Processing a mixture of in-vitro-derived MKs and PLTs on the adapted device yielded a pure PLT population and did not induce PLT pre-activation. MKs recovered from the separation process were unaffected with respect to viability and ploidy, and were able to generate PLTs after reseeding in culture. Being able to efficiently harvest in-vitro-derived PLTs brings this technology one step closer to clinical relevance.

Megakaryocyte polyploidization and proplatelet formation in low-attachment conditions

“…To determine if ULA-like culture would affect primary human MK maturation and PPF, mPB CD34 + HSPCs from 2 different donors were first differentiated to MKs on a TC surface, as previously described [37]. On day 7, the culture was selected for CD41 + MKs, which were then reseeded on either a TC or polyHEMA-coated surface in the presence of Nic.…”

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies

“…Different 3D tools have been described thus far in the attempt of supporting thrombopoiesis by both mouse and human Mks differentiated from either hematopoietic progenitors or the promising induced pluripotent stem cells. 4,5,[55][56][57][58][59] Despite the usefulness for the advancement in providing systems for platelet production, all these models did not reproduce the complex microenvironment where megakaryopoiesis physiologically takes place. In this regard, a fundamental advantage of our model is its ability to provide the first ex vivo tissue system able to reproduce the 3D structure of the bone marrow niche, in combination with ECM components and physiologic shear rate, for the study of Mk function and platelet formation.…”

Understanding platelet generation from megakaryocytes: implications for in vitro–derived platelets

“…21,[38][39][40] The effects of modulating and/or inhibiting this pathway to increase megakaryocyte numbers should be further explored to optimize maturation of undamaged megakaryocyte populations. One study showed that the overexpression of Bcl-xL in human megakaryocytes increased proliferative capacity and survival.…”