Harvest of adherend cells from Cytodex® 1 beads

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 TrypLE^TM 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.

Steps

sampling

withdraw homogenized cell solution from culture vessel

and transfer to centrifuge tube 10-50mL

1.1.

take a sample from each tube for counting to determine available cell number

Micro carrier solving

add 0.1% volume of dextranase (v/v)

transfer samples to a tube rotator and place in an incubator 10rpm,37°C

take out tubes and centrifuge 600x g

carefully discard supernatant

single cells

add a small volume 1-5mL of 2x TrypLE^TM and completely solve the pallet

6.1.

add more 9-45mL of 2x TrypLE^TM, transfer tubes to a Tube rotator and place in an incubator 10rpm,37°C

take out tubes and centrifuge 600x g

discard supernatent and solve pallet in adequat amount of culture medium 1-5mL

8.1.

filtrate cell solution through a 40 µm Filter to exclude micro carrier fragments

Yield, viability and follow up

count cells and determine viability

10.

use cells for further analysis