Origin and Features of the hBM-MSCs
Human Bone Marron Mesenchymal Stem Cells (BMSCs) are isolated from bone marrow. Bone marrow is the hematopoietic tissue of the body, located within the cavities of many bones. In adults, there are two types of bone marrow: red marrow and yellow marrow. Red marrow produces red blood cells, platelets, and various types of white blood cells. Platelets are involved in hemostasis, while white blood cells destroy and inhibit various pathogens, including bacteria and viruses; certain lymphocytes can produce antibodies. Therefore, bone marrow is not only a hematopoietic organ but also an important immune organ.
Bone marrow resides in the medullary cavities of long bones (such as the humerus and femur) and the intertrabecular spaces of cancellous bone in flat bones (such as the ilium). It is a spongy tissue. The bone marrow capable of producing blood cells is somewhat red and is called red marrow. At birth, red marrow fills all marrow cavities. With age, the number of fat cells increases, and a significant portion of the red marrow is replaced by yellow marrow. Eventually, red marrow is almost exclusively found in the marrow cavities of flat bones.
Residing within the bone marrow stromal system are Bone Marrow Mesenchymal Stem Cells (BMSCs). These are stem cells, distinct from hematopoietic stem cells, possessing high self-renewal capacity and multidirectional differentiation potential. They can differentiate into various tissues such as bone, cartilage, muscle, skin, fat, and nerve tissue, thus serving as seed cells in tissue engineering. In bone marrow, BMSCs constitute a very low proportion, approximately 0.001% to 0.1%, of the total nucleated cells.
The in vitro culture requirements for BMSCs are relatively demanding. During the cultivation process, they are influenced by factors such as adherence time, seeding density, serum concentration, incubation temperature, and the pH of the culture medium.
Morphological Observation and Live Cell Station Detection of hBM-MSCs at Different Densities
Figure 1. hBM-MSCs cultured on Ucallm® Ultra-Low Attachment Surface forms tumor spheroids. hBM-MSCs were planted in 96-well Ultra-Low Attachment Surface at concentrations of 500, 1000, 2000, 4000, and 8000 cells per well. Live-cell imaging was conducted at 24, 48, 72, 96, and 120 hours after seeding. Scale bars represent 200 μm.
Method
Culture Conditions
hBC-MSCs: 93% MSCM + 5% FBS +1%P/S + 1% MSCGS
Cell recovery
1) Frozen vials of hBC-MSCs were retrieved from liquid nitrogen and rapidly thawed in a 37 ℃ water bath with gentle agitation.
2) The thawed cell suspension was transferred into a centrifuge tube containing 3 mL of culture medium and centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was discarded.
3) The cell pellet was resuspended in complete medium, seeded into a culture dish, and gently mixed. Cells were cultured in a standard incubator at 37 ℃ with 21% O₂ and 5% CO₂.
Cell Passaging
1) Cells were passaged when reaching 80% confluence.
2) The culture medium was aspirated, and the cells were washed once with PBS.
3) 1–2 mL of 0.25% trypsin was added to digest the cells at 37 ℃ for 1–3 minutes. Digestion was considered complete when cells became rounded and detached under microscopic observation.
4) Trypsin was quickly removed, and complete medium was added to neutralize the enzyme. The cells were gently pipetted to form a single‑cell suspension and subcultured at a split ratio of 1:2 to 1:4. Expanded culture was continued under the same conditions (37 ℃, 21% O₂, 5% CO₂).
Cell Seeding
hBC-MSCs in the logarithmic growth phase with good viability were harvested and seeded into a 96‑well U‑bottom ultra‑low attachment plate at densities of 1000, 2000, 4000, and 8000 cells per well (one plate was prepared). Each density was replicated in five wells. The peripheral wells of the plate were filled with 100 μL of sterile PBS. The plate was then transferred to a live‑cell imaging station for culture and imaging.
Note: The live‑cell station, pre‑installed in a CO₂ incubator (Thermo, 3111), was pre‑warmed for 30 minutes and maintained at 37 ℃ with 21 % O₂, 5 % CO₂, and saturated humidity.
Medium Change
The medium for hBC-MSCs was replaced every 24 hours (including those in the live‑cell station). The frequency of medium changes varied with cell density: wells seeded with 1000 and 2000 cells per well were changed less frequently than those with 4000 and 8000 cells per well. This process was continued for 120 hours, totaling five medium changes. During each change, the old medium was carefully aspirated from all five replicate wells of a given density at once, and promptly replaced with 100 μL per well of fresh complete medium.
Materials and Instruments
Table 1 Main equipment
Name | Manufacturer | Catalog Number |
CO2 Incubator | Thermo | 3111 |
Inverted Microscope | OLYMPUS | IX73 |
96-well Ultra-Low Attachment（U-bottom）Cell Culture Plate | Ucallm | L1096UA |
Table 2 Major Reagents
Name | Manufacturer | Catalog Number |
hBM-MSCs | ATCC | PCS-500-012 |
MSCM（With MSCGS） | Sciencell | 7501 |
FBS | Gibco | 10099141 |
Penicillin-Streptomycin Solution | Gibco | 15140122 |
0.25% Trypsin | Gibco | 25200072 |
PBS Buffer Solution | Gibco | 10010023 |
References
[1] Ferreira LP, Gaspar VM, Monteiro MV, Freitas B, Silva NJO, Mano JF. Screening of dual chemo-photothermal cellular nanotherapies in organotypic breast cancer 3D spheroids. J Control Release. 2021 Mar 10;331:85-102.
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