Abstract

There is a high demand in various fields to develop complex cell cultures. Apart from titer plates, Transwell inserts are the most popular device because they are commercially available, easy to use, and versatile. While Transwell inserts are standardized, there are potential gains to customize inserts in terms of the number of layers, height between the layers and the size and composition of the bioactive membrane. To demonstrate such customization, we present a small library of 3D-printed inserts and a robust method to functionalize the inserts with hydrogel and synthetic membrane materials. The library consists of 24- to 96-well sized inserts as whole plates, strips, and singlets. The density of cultures (the number of wells per plate) and the number of layers was decided by the wall thickness, the capillary forces between the layers and the ability to support fluid operations. The highest density for a two-layer culture was 48-well plate format because the corresponding 96-well format could not support fluidic operations. The bottom apertures were functionalized with hydrogels using a new high-throughput dip-casting technique. This yielded well-defined hydrogel membranes in the apertures with a thickness of about 500 µm and a %CV (coefficient of variance) of < 10%. Consistent intestine barrier was formed on the gelatin over 3-weeks period. Furthermore, mouse intestinal organoid development was compared on hydrogel and synthetic filters glued to the bottom of the 3D-printed inserts. Condensation was most pronounced in inserts with filters followed by the gelatin membrane and the control, which were organoids cultured at the bottom of a titer plate well. This showed that the bottom of an insert should be chosen based on the application. All the inserts were fabricated using an easy-to-use stereolithography (SLA) printer commonly used for dentistry and surgical applications. Therefore, on demand printing of the customized inserts is realistic in many laboratory settings.

Highlights

  • There is a high demand in various fields to develop complex cell cultures

  • We considered the wall thickness, the wall-to-wall distance, and the number of layers that could be stacked in the respective well format

  • We present a small library of inserts that challenge the physical limits of 3D printing, capillary effects, and liquid handling

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Summary

Introduction

There is a high demand in various fields to develop complex cell cultures. Apart from titer plates, Transwell inserts are the most popular device because they are commercially available, easy to use, and versatile. While Transwell inserts are standardized, there are potential gains to customize inserts in terms of the number of layers, height between the layers and the size and composition of the bioactive membrane. Transwell hanging inserts have been extensively used to recreate single barrier tissues like the intestine, skin, and blood–brain-barrier They are used to interconnect tissues by co-culturing multiple cell lines or creating 3D tissue such as organoid cultures with ­hydrogels[8–10]. The drawbacks of commercial Transwell inserts are the lack of flexibility to choose the material and size of the bioactive cell culture area. The SLA and projection printers (e.g., digital light processing (DLP) printers) have better resolution and provide liquid-proof ­prints[14] In these printers, light is utilized to crosslink a photo-curable resin that is biocompatible for cell culture a­ pplications[14,15]. This property allows for defining the bioactive area with a filter or a hydrogel to resize and shape the cell culture area according to experimental needs

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