Cell seeding protocol for 3D silk scaffolds (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Sumit Murab; David Kaplan; Sophia U. Szymkowiak

Keywords

[‘3D culture’, ‘culture’]

Subjects

[‘Cell culture’, ‘Tissue culture’]

Release Date

2017-06-27

Abstract

The current protocol describes the methods for seeding cells into 3D silk scaffolds. Subsections of the protocol describe seeding directly into silk scaffolds, seeding after coating the scaffolds with ECM, or encapsulating cells in ECM within the silk scaffolds.

Reagents

1. Silk lyophilized scaffolds i. Silk scaffolds with interconnected porous structure made by controlled lyophilization. Typical pore size ~ 90 µm, but can be altered during fabrication, as needed from ~25 up to ~150 um (larger sized interconnect pores, up to ~1,000 um, can be generated using porogen leached silk scaffolds) ii. Cut the scaffolds into discs with 6 mm diameter (with a biopsy punch) and sliced 250 µm–1 mm thick to fit in the bottom of a 96-well plate (or any other workable shape/thickness, as needed)
2. Cell culture media (cell specific)
3. 1X PBS
4. 0.5 % Trypsin (or other cell dissociation reagent)

Procedure

I. Seeding cells directly in silk

Preparing scaffolds for cell seeding i. Sterilize scaffolds by soaking in 70% ethanol, then remove and aspirate to dry (alternatively, the scaffolds can be autoclaved or treated by ethylene oxide for sterilization) ii. Wash scaffolds several times in PBS to remove residual ethanol iii. Aspirate scaffolds (to near dryness) with an aspirating pipette iv. Soak scaffolds in cell media, incubate at 37°C for at least 1 hour v. Remove excess media by aspiration (to near dryness) and seed cells (see below)

Cell seeding i. Prepare cell suspension in media at desired concentration (for RPTEC/Tert1 cells in 96 well plates we typically resuspend cells at 10,000 cells/μL and add 25 μL/1mm scaffold) ii. Add cell suspension onto the dry scaffold and press the scaffold evenly with a micro-pipette tip (P-200) to promote even distribution of the cells within the bulk (pipette out any excess media not soaked by the scaffold back onto the top and continue pressing with the tip until full volume is absorbed) iii. Place in the incubator for ~1 hour, then flip the scaffolds over and incubate for an additional 1 hour iv. Gently add enough media to fully cover scaffolds (150μL media/well in a 96-well plate) v. Following overnight incubation, move the scaffolds to new wells and continue culturing

Alternative: If scaffold number is very high or uniform cell seeding is difficult to achieve, plates can be centrifuged at a low speed after addition of cells to the top of the scaffold to promote homogenous distribution of cells throughout the scaffolds

II. Coating silk scaffolds with ECM

Preparing scaffolds for ECM coating i. Aspirate scaffolds (to near dryness) and add desired volume of (cold) ECM ii. Press scaffolds evenly with a micro-pipette tip (P-200) for even distribution of ECM within the bulk iii. Place in the tissue culture incubator for 30 minutes or until full ECM gelation

Cell seeding i. Use aspirating pipette to remove excess ECM from scaffold pores ii. Proceed to cell seeding (as described in Section I)

III. Seeding cells (resuspended in ECM) in silk scaffolds

Preparing scaffolds for ECM coating i. Prepare scaffolds for cell seeding as described previously (as described in Section I) ii. Dry scaffolds using aspirating pipette

Cell seeding i. Resuspend cells in cold ECM to desired concentration, on ice ii. Aspirate scaffolds (to near dryness) iii. Follow cell seeding procedure to seed cells onto scaffolds (as described in Section I)

Consortium

(Re)Building a Kidney (RBK) Consortium