Table 2.
Summary of critical steps for BMAd and BMSC isolation to highlight differences between protocols.
| Main steps | EDIN | TOUL | LILL | LAUS | PRAG |
|---|---|---|---|---|---|
| Material | |||||
| Bone site of sampling | Femoral proximal metaphysis and diaphysis Femoral head |
Femoral proximal metaphysis and diaphysis | Trabecular bone from distal femoral epiphysis | Femoral head (epiphysis + variable quantity of metaphysis) Iliac crest |
Iliac crest |
| Sample type | BM aspirates | BM aspirates | Cancellous bone | BM aspirates | BM aspirates |
| Final material obtained | BMAds | BMAds | BMAds | BMSCs and BMAd-enriched fraction | BMSCs and BMAd-enriched fraction |
| Digestion | |||||
| Buffer | KRH, 5.5 mM glucose 3% BSA |
PBS 2% BSA |
DMEM, 5.5 mM glucose 3% BSA |
DMEM, 10% FBS | - |
| Collagenase type, manufacturer (reference number) | Type 1, Worthington Biochemicals (LS004196) | from C. histolyticum, Sigma Aldrich (C6885) | NB 4 standard grade, SERVA Electrophoresis (17454) | Type I, Gibco (#17100-017) | - |
| Collagenase concentration | 250 UI/mL | 0.2 to 0.3 UI/mL | 1 UI/ml | - | |
| Digestion time | 45min | Max. 20 min | 15-30 min | 45 min -1h | - |
| Washing | |||||
| Buffer | KRBH 5.5 mM glucose | KRBH 0.5% BSA |
DMEM, 5.5 mM glucose/ 3% BSA |
PBS/1% BSA | PBS/1% BSA |
Different protocols have been set up to isolate BMAds (from 3 institutes: EDIN, TOUL, LILL) or BMSCs (from 2 institutes: LAUS, PRAG). Differences related to biological material, digestion and washing parameters are highlighted.
Abbrevations referring to institute cities of members within BMAS Biobanking WG. EDIN: Edinburgh (WC); TOUL: Toulouse (CA); LILL: Lille (SL), LAUS: Lausanne (ON); PRAG: Prague (MT).