Sep 12, 2023
Harvest of adherend cells from Cytodex® 1 beads
- David König1,
- Kim Kremser1,
- Sina Bartfeld1,2,
- Shirin Kadler2
- 1Medical Biotechnology, Technische Universität Berlin, Berlin, Germany;
- 2Si-M / “Der Simulierte Mensch” a science framework of Technische Universität Berlin and Charité - Universitätsmedizin Berlin, Berlin, Germany
Protocol Citation: David König, Kim Kremser, Sina Bartfeld, Shirin Kadler 2023. Harvest of adherend cells from Cytodex® 1 beads. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4w7pgmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 13, 2023
Last Modified: September 12, 2023
Protocol Integer ID: 78658
Keywords: micro carrier, Cytodex1, adherent cells, MSC, Chondrocytes, Fibroblasts, dextranase, cell culture, dextran, cell harvest
Funders Acknowledgement:
ZIM Funding Project - Federal Ministry For Economic Affairs And Climate Action
Grant ID: KK5145501AD0
Abstract
Expanding adherend cells to quanteties suitable for cell therapy is a subject of increasing relevance.
Variouse cell types either autolog or allogen - could in future be used for medical purposes / in clinical application as a key component in therapy. This protocol discribes how to harvest adherend cells from dextran based Cytodex® 1 beads after expansion. Since other enzymatic schemes like Trypsin-EDTA or TrypLETM have shown low efficiency and yield - here we present an approach that is easy in implementation and delivers cells with high harvesting yield and high viability.
Guidelines
This protocol can surely be further improved but besides that is pretty much strait forward - crutial steps are the centrifugation and supernatent discartion where many cells can be lost if samples are not handled with due care.
Materials
DMEM - 4.5g/L glucose - stable glutamine - sodium pyrovate - Biowest [L0103-500]
10% FBS - Corning
1x P/S - Corning
PBS
10x TrypLE Express - Gibco
Dextranase Plus L, 1,6-α-D-Glucan-6-glucanohydrolase from Chaetomium erraticum
Novozymes - >100 KDU-A/G [Dextranase units]
Cytiva - Cytodex1 beads [17044803]
Clean bench
Incubator - 37°C - 5% CO2
Centrifuge
Centrifugation tubes 15/50mL
Eppendorf tubes 1.5mL
Tube rotator
Pipetboy + pipets [5-50mL]
Pipets - [10-1000uL]
NucleoCounter NC-200 + Solution A100 and B
Neubauer Chamber
Vacuum pump
Spinner Flask - Pfeiffer 500mL
Magnetic stirrer - Technomara
adherent primary cells - MSC / Chondrocytes / HUVEC / Fibroblasts
Safety warnings
There are no particular safety risks exceeding the risk during cell culture.
sampling
sampling
1h 26m
1h 26m
withdraw homogenized cell solution from culture vessel
and transfer to centrifuge tube 10-50 mL
take a sample from each tube for counting to determine available cell number
Micro carrier solving
Micro carrier solving
1h 26m
1h 26m
add 0.1 % volume of dextranase (v/v)
transfer samples to a tube rotator and place in an incubator 10 rpm, 37°C, 01:00:00
1h
take out tubes and centrifuge 600 x g, 00:08:00 , acc 9 - dec 5
8m
carefully discard supernatant
single cells
single cells
1h 26m
1h 26m
add a small volume 1-5 mL of 2x TrypLETM and completely solve the pallet
add more 9-45 mL of 2x TrypLETM, transfer tubes to a Tube rotator and place in an incubator 10 rpm, 37°C, 00:10:00
10m
take out tubes and centrifuge 600 x g, 00:08:00 , acc 9 - dec 5
8m
discard supernatent and solve pallet in adequat amount of culture medium 1-5 mL
filtrate cell solution through a 40 µm Filter to exclude micro carrier fragments
Yield, viability and follow up
Yield, viability and follow up
count cells and determine viability
use cells for further analysis