Ref No: 006005004140
Date: 16-JAN-2023
Author: PK,SN. Version: 2

CELL RECOVERY PROTOCOL

Cell Collect A
This is a suggested procedure, please adjust according to your experimental needs.
To maintain the sterility of the product, work under sterile conditions.

Protocol aim

Materials neededThis protocol provides instructions for the use of Cell
Collect A, an enzyme-based proprietary formulation,
to enhance cell isolation or reduce viscosity ofalginate-based bioinks. The proposed method allows
for digesting of bioinks either fully or partially andharvesting cells that can subsequently be used forcell viability analyses, RNA isolation, protein
extraction, western blots, and qPCR among otherdownstream applications. Under sterile conditions,recovered cells can be re-plated for culture or usedfor analysis.

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Cell Collect A*PBS (1X)
Cell-laden bioprinted constructs
Cell shaker
1 M NaOH (optional)
Cell culture media (optional)
Cell strainer (40-70 µm nylon)
Centrifuge tubes*The product can be purchased in the CELLINKshop at www.cellink.com/shop.

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Protocol1.

Diluting Cell Collect A (optional)

MATERIAL
Cell Collect A
PBS (1X)DESCRIPTION•

Dilute Cell Collect A in PBS to achieve desired concentration. See Table 1 for recommended
concentrations.

Table 1. Recommended Cell Collect A concentrations for partial or complete digestion of bioink droplets.
Digestion

Cell Collect A dilution

Bioink droplet volume (µL)

Minimum time to digest
at 4°C (min)Partial**Diluted more than twice

10-100

15–30

10-25
15–30
25-100***30–60**Partial bioink digestion may be desired for improved proliferation, porosity or delivery of small molecules. For this,diluted concentrations of Cell Collect A can be added to constructs. Adjust dilution according to application.
***For bioprinted constructs larger than 100 µL longer incubation times may be required.
Complete2.

Non-diluted

Dissociation

MATERIAL
Cell-laden bioprinted constructs
Cell shaker
NaOH (optional)
PBS (1X) (optional)
Cell culture media (optional)

DESCRIPTION•••

Remove the cell culture medium from construct(s).
Add 10 times as much volume of Cell Collect A as the volume of bioink construct to desired well(s).
Make sure that the solution covers the construct.
Place the entire well plate on a cell shaker at 4°C for ~30 min, or until fully dissolved, see Table 1
recommended incubation times.

Note: Gentle disintegration with a large orifice pipette tip is required to fully break down constructs.
•For partial degradation, the degradation process can be inhibited after the desired degradation time byadding 1 M NaOH in a 1:10 volume ratio to initially added Cell Collect A, waiting for 15 min, washingonce and then carefully replacing it with fresh media.

Note: To slow cell signalling pathways, it’s recommended to use Cell Collect A at 4°C. If desired, 3D
constructs in Cell Collect A can be incubated at room temperature or 37°C.

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3.

Cell isolation

MATERIAL
Cell strainer
Centrifuge tube(s)PBS (1X)DESCRIPTION••

4.

Place a cell strainer over a centrifuge tube and wet the bottom of the strainer with sterile PBS tofacilitate flow-through.
In the same tube, use the cell strainer to filter the dissolved ECM-cell suspension.

Centrifuge

DESCRIPTION•••

Centrifuge the collected cell suspension at 200 g for 3-4 min.
Remove supernatant.
Cell pellet is ready to use for desired applications.

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