Cryomilling of Decellularized Tissue: A Technique to Obtain Fine Extracellular Matrix Powder
Published: April 30, 2023
Abstract
Source: Kornmuller, A. et al. Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms. J. Vis. Exp. (2017)
In this video, we present the protocol to obtain finely ground extracellular matrix powder from lyophilized decellularized tissue by cryomilling. The powdered ECM retains its biochemical composition and can be used for microcarrier synthesis.
Protocol
1. Decellularized Tissue Processing Decellularization and Lyophilization Decellularize tissue(s) of interest following an established protocol. NOTE: Scaffolds in the current study have been prepared following published decellularization protocols for human DAT, porcine DDT, and porcine DLV. Commercially available, insoluble collagen can also be used to fabricate foams and microcarriers, such as collagen sourced from bovine tendon, which has been used succ…
Declarações
The authors have nothing to disclose.
Materials
| Alligator clip leads | VWR | 470149-728 | |
| Aluminium foil | Fisher Scientific | 01-213-101 | |
| Analytical balance | Sartorius | CPA225D | |
| Cryomilling system | Retsch | 20.745.0001 | MM 400 |
| Dessicator | Fisher Scientific | 8624426 | For lyophilized ECM and bioscaffold storage |
| Dewar flask | Fisher Scientific | 10-196-6 | Low form; volume range of 250 – 500 mL |
| Double distilled water (ddH2O) | From Barnstead GenPure xCAD Water Purification System | ||
| ECM (Extracellular Matrix) | Isolated from human adipose tissue, porcine dermis or porcine myocardium, as described in Flynn et al. 2010, Reing et al. 2010, and Wainwright et al. 2010 | ||
| Forceps | VWR | 37-501-32 | For transferring the foams |
| Freezer (-20 °C) | VWR | 97043-346 | |
| Freezer ( -80 °C) | Thermo Scientific | EXF40086A | |
| Glass vials | Fisher Scientific | 03-339-26D | To store lyophilized cryomilled ECM |
| Liquid nitrogen | For electrospraying | ||
| Lyophilizer | Labconco | 7750021 | FreeZone4.5 |
| Milling chamber | Retsch | 02.462.0213 | 25 mL volume recommended |
| Milling balls | VWR | 16003-606 | 10 mm diameter, stainless steel recommended |
| Penicillin-streptomycin | Life Technologies | 15140-122 | Used for proliferation media |
| Pipet-Aid XP | Mandel Scientific | DRU-4-000-101 | |
| Scoopula | VWR | 89259-968 | For collecting microcarriers |
| Surgical scissors | VWR | 82027-590 |
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Cryomilling of Decellularized Tissue: A Technique to Obtain Fine Extracellular Matrix Powder. J. Vis. Exp. (Pending Publication), e20483, doi: (2023).