1. Objective
This protocol describes the procedure of placing and culturing tissue fragments (e.g., PDX (patient-derived xenograft) materials, spheroids, or organoids) in the OrganoPlate® Graft using tip-based liquid handlers.
2. Background
The OrganoPlate® Graft (see figure 1) can be used to grow vascular tubules that resemble blood vessels in vivo by seeding endothelial cells in the perfusion channels. When the endothelial tubules are formed, pro-angiogenic factors can be added to trigger the formation of angiogenic sprouts, resulting in a vascular network (see Figure 2). This protocol describes the procedure of placing tissue fragments in the gel chamber of the OrganoPlate® Graft using an automated setting.
3. Materials
• OrganoPlate® Graft (MIMETAS, 6401-400-B) with or without a vascular network
o The procedure for formation of a vascular network is described in the following protocol: OrganoPlate® Graft Vascular Network Formation
• Tissue for placement i.e., spheroids, organoids, tumor tissue, or cell aggregates
• Medium for the endothelial cells: 14 mL for all the medium in- and outlets
• Tissue-specific medium: 4mL for all the graft chambers
• Tip-based liquid handling systems
• Disposable wide-bore pipette tips
o The use of wide-bore tips is highly recommended. Only with very small tissues, the regular tips might be compatible.
4. Procedures
4.1 Robotic system preparation
1. Familiarize yourself with the OrganoPlate® Graft plate and chip layout
2. Set the OrganoPlate® Graft dimensions into your robotic system (see below for OrganoPlate® Graft dimensions)
3. If necessary, test the robotic system with an empty OrganoPlate® Graft
4.2 Tissue placement
This protocol describes the procedure for tissue placement that does not require ECM embedding (see figure 3).
Medium change
1. Before placement of tissue, make sure all the medium in the in- and outlets and graft chambers are filled with fresh medium
a) Aspirate the media from all perfusion inlet and outlet wells and Graft chambers
b) Add 50 μL of endothelial-specific medium to the inlet and outlet well of the perfusion channels
c) Add 50 μL tissue-specific medium in the Graft chambers
o Medium change can be done with a manual pipette or with the robotic system
Tissue placement
1. Aspirate the tissue material in a volume of 5-50μL with the wide-bore tip
2. Allow the tissue to settle at the bottom of the pipette tip
o The speed of the settlement depends on the volume and the size of the tissue
3. Position the pipette tip with the tissue in the middle of the graft chamber well. Move the pipette tip towards the graft chamber hole at a fast pace to prevent the tissue to float out of the tip before the graft chamber hole has been reached.
a. Make sure to not disrupt the ECM by moving the pipette tip too deep
b. Try to position the pipette tip right above the graft chamber hole (see OrganoPlate® Graft dimensions below for well depth)
4. When the pipette tip enters the graft chamber well, leave it there for 2-3 seconds. Allow the tissue to flow out of the wide board tip and position on the graft chamber hole
a. The placement of the tissue is gravity-based, make sure not to dispense in the graft chamber hole
5. Retract the pipette tip slowly from the graft chamber to avoid displacement of the freshly placed tissue
6. Repeat steps 2-6 for all the chips in the OrganoPlate® Graft
a. If a multichannel head (i.e., 8-, 96- or 384-well) is available on the robotic system, the placement of the tissues can be done using multiple tips