Stem Cell Therapy Cuts Close to the Bone

Cryopreserving whole bone fragments rather than single cells lets scientists save stem cell rich bone marrow with minimal tissue processing. Image credit:

Scientists in Modena, Italy have just published an article describing an optimized method for cryopreserving whole bone fragments [1]. The article is generating keen interest among stem cell scientists, who are often faced with a conundrum on the best way to preserve mesenchymal stem cells for which the downstream applications are still undecided.

Current methods for banking human bone marrow-derived mesenchymal stem cells (hBM-MSCs) involves isolating and culturing the cells, an expensive and time-consuming procedure. Isolated cells are often cultured in animal-derived serum and supplements, which can trigger severe immune response or infections when the cells are administered to patients.

To avoid these issues, the authors proposed to work out a method for storing the stem cells without culturing them–instead, they wanted to be able to freeze entire bone fragments. Stem cells could then be generated at need, and handled in a manner befitting their intended use.

“Trabecular” bone is the spongy bone found at the ends of long bones, or in the ribs, skull, or spinal vertebrae. It consists of a loose latticework of bone filled with connective tissue and stem cell rich bone marrow. Cryopreserving whole trabecular fragments containing bone marrow is both faster and less expensive than isolating hBM-MSCs. Contamination risks during tissue culture are avoided, and when banking tissue samples with no requirement for immediate isolation, the proposed method provides flexibility in downstream clinical uses.

Having decided on a course of action, the research group’s real question became whether the stem cells preserved within frozen bone fragments were the functional equivalent of freshly isolated hBM-MSCs. Previous studies suggested that cryopreservation of hBM-MSCs in freezing medium containing a high concentration of human serum, in combination with low levels of cryoprotectants (mostly DMSO), would keep viability rates high, while adhering to GMP guidelines for human stem cell therapy. It’s wise to meet GMP standards as early as possible in your cryopreservation protocols, to avoid expensive delays when treatments are ready to move forward to clinical trial. Human serum was accordingly obtained from the blood of healthy donors, and used to cryopreserve bone specimens collected from orthopedic surgery patients who agreed to participate in the study.

Bone fragment specimens cryopreserved with the newly designed human serum based protocol were stored in liquid nitrogen for a year, prior to being tested. Human BM-MSCs isolated from the cryopreserved fragments performed well in viability assays, stem cell marker assays, and proliferation assays. They also exhibited classic stem cell morphology, the ability to initiate form colony–forming units, and retained the capacity to differentiate into osteogenic, adipogenic, and myogenic cell lines. For each of the examined criteria, hBM-MSCs isolated from cryopreserved bone fragments performed just as well as freshly isolated hBM-MSCs.

The authors concluded that the new cryopreservation method will allow for clinical application of stem cells cryopreserved with GMP compatible protocols. It should also cut expenses, limit contamination risk, and in general facilitate human stem cell banking for clinical use.

[1] Carnevale G., et al.Optimized Cryopreservation and Banking of Human Bone-Marrow Fragments and Stem Cells. Biopreservation and Biobanking. 14 (2): 138-148. 2016.