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. 2018 Feb;18(2):132-139.
doi: 10.1002/elsc.201700008. Epub 2017 Nov 24.

A modified 384-well-device for versatile use in 3D cancer cell (co-)cultivation and screening for investigations of tumor biology in vitro

Miriam Widder  1 , Karen Lemke  1 , Bünyamin Kekeç  1 , Tobias Förster  1 , Andreas Grodrian  1 , Gunter Gastrock  1
Affiliations

A modified 384-well-device for versatile use in 3D cancer cell (co-)cultivation and screening for investigations of tumor biology in vitro

Miriam Widder et al. Eng Life Sci. 2018 Feb.

Abstract

Pancreatic cancer exhibits a worst prognosis owed to an aggressive tumor progression i.a. driven by chemoresistance or tumor-stroma-interactions. The identification of candidate genes, which promote this progression, can lead to new therapeutic _targets and might improve patient's outcome. The identification of these candidates in a plethora of genes requires suitable screening protocols. The aim of the present study was to establish a universally usable device which ensures versatile cultivation, screening and handling protocols of cancer cells with the 3D spheroid model, an approved model to study tumor biology. By surface modification and alternative handling of a commercial 384-well plate, a modified device enabling (i) 3D cultivation either by liquid overlay or by a modified hanging drop method for (ii) screening of substances as well as for tumor-stroma-interactions (iii) either with manual or automated handling was established. The here presented preliminary results of cell line dependent dose-response-relations and a stromal-induced spheroid-formation of the pancreatic cancer cells demonstrate the proof-of-principle of the versatile functionality of this device. By adapting the protocols to automation, a higher reproducibility and the ability for high-throughput analyses were ensured.

Keywords: 3D cell culture; Automation; Pancreatic cancer; Substance screening; Tumor‐stroma‐interaction.

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Figures

Figure 1
Figure 1
Schematic drawing of experimental procedure for LO‐ and MHD‐method within the modified 384‐well‐device. Here, steps depicted with * were automatically performed (A). Valuation of the modified 384‐well‐device by specifying its advantages and disadvantages (B).
Figure 2
Figure 2
Suitability of the modified 384‐well‐device for substance screenings. Scheme and photo documentation of PaTu‐8988t wildtype cells cultivated 48 h with LO‐ (A) and MHD‐method (B). Dose‐response‐relations and calculated IC50‐values of PaTu‐8988t wildtype cells treated with gemcitabine. Several independent approaches (n) were performed manually with LO‐method (□, n = 7) or automatically with LO‐ (◇, n = 6) or MHD‐method (△, n = 5). Manually performed analyses with 2D cultures (×, n = 2) serve as control (C). Automated microscopic determination of HT29‐cells cultivated 2D or 3D with LO‐ and MHD‐method and calculated IC50‐values from these cultures treated with paclitaxel in two independent approaches (D). Dose‐response‐relation and calculated IC50‐values of two doxycycline‐inducible mutants from PaTu‐8988t: clone I (E) and clone II (F) treated with gemcitabine in two independent approaches (LO‐method automatically ▴ and manually •). Data are depicted as mean +/‐ SD with 12 samples contributing to each concentration of every individual approach n. Bar – 500 μm.
Figure 3
Figure 3
Investigation of tumor‐stroma‐interactions within the modified 384‐well device. Microscopic determination of PaTu‐8988t wildtype cells mixed with HPSC in different ratios seeded w/wo doxycycline either with LO‐ or MHD‐method and cultivated for 48 h (A). Microscopic determination of two doxycycline inducible mutants from PaTu‐8988t and Suit2‐007 wildtype cells mixed with HPSC in different ratios seeded with doxycycline in LO‐method and cultivated for 48 h (B). Bar – 500 μm.
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