Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Summary

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Abstract

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.

Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.

The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Introduction

Spheroids are considered a scaffold-free approach in tissue engineering. ASCs are capable of forming spheroids by the self-assembly process. The spheroid's 3D microarchitecture increases the regenerative potential of ASCs, including the differentiation capacity into multiple lineages^1,2,3. This research group has been working with ASC spheroids for cartilage and bone tissue engineering^4,5,6. More importantly, spheroids are considered building-blocks in the biofabrication of tissues and organs, mainly due to their fusion capacity.

The use of spheroids for tissue formation depends on three main points: (1) the development of standardized and scalable robotic methods for their biofabrication⁷, (2) the systematic phenotyping of tissue spheroids⁸, (3) the development of methods for the assembly of 3D tissues⁹. These spheroids can be formed with different cell types and obtained through various methods, including hanging drop, reaggregation, microfluidics, and micromolds^8,9,10. Each of these methods has advantages and disadvantages related to the homogeneity of size and shape of the spheroids, recovery of the spheroids after formation, the number of spheroids produced, process automation, labor intensity, and costs¹¹.

In the micromold method, the cells are dispensed and deposited at the bottom of the micromold because of gravity. The non-adhesive hydrogel does not allow the cells to adhere to the bottom, and cell-to-cell interactions lead to the formation of a single spheroid per recession^8,12. This biofabrication method generates spheroids of homogeneous and controlled size, can be robotized for large-scale production in a time-efficient manner with minimal effort, and has good cost-effectiveness-critical factors in the design of a biofabrication of tissue spheroid^7,8. This method can be applied to form spheroids of any cell lineage to prepare a new tissue type with predictable, optimal, and controllable characteristics⁸.

Biofabrication is defined as "the automated generation of biologically functional products with structural organization ..."¹³. Therefore, the automated production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the bioprinted tissue by spheroid fusion. In this study, to improve the scalability of ASC spheroid biofabrication, we use an automated pipetting system to seed the cell suspension, thus ensuring the homogeneity of spheroid size and shape. This paper shows that it was possible to produce a large number (thousands) of spheroids needed for 3D bioprinting approaches to biofabricate more complex tissue models.

Protocol

The ASCs used in this study were previously isolated from healthy human donors and cryopreserved as described¹⁴ according to the Research Ethics Committee of Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Brazil (25818719.4.0000.5257). See Table of Materials for details regarding all the materials and equipment used in this study.

1. Trypsinization of ASC monolayer at passage three

1. Open the tissue culture 175 cm² flask containing the monolayer of ASCs at 80% confluence and discard the supernatant.
2. Add 7 mL of phosphate-buffered saline (PBS, 0.01 M) and wash the monolayer twice. Next, discard the liquid.
3. Add 5 mL of 0.125% trypsin with 0.78 mM ethylenediaminetetraacetic acid (EDTA). Next, incubate for 5 min at 37 °C.
4. Add 10 mL of low-glucose Dulbecco's Modified Eagle Medium (DMEM LOW) with 10% fetal bovine serum (FBS) to the tissue culture 175 cm² flask and mix the cell suspension well with a 10 mL sorologic pipette.
5. Harvest the cell suspension with a 10 mL serological pipette and transfer it to a 50 mL centrifuge tube. Next, centrifuge at 400 × g for 5 min to obtain the pellet of ASCs. Resuspend the cell pellet using DMEM LOW containing 10% FBS.
6. Perform a cell count by trypan exclusion.
7. After counting, take a total of 1 × 10⁶ ASCs in a separate 15 mL centrifuge tube to seed the 81 recessions micromolded with non-adherent hydrogel (a total of 10,000 cells/mL seeded here). To seed 256 recessions of micromolded non-adherent hydrogel, take a total of 5 × 10⁵ ASCs into a centrifuge tube (a total of 2,500 cells/mL seeded here).

2. Micromolded non-adherent agarose hydrogel fabrication

1. Prepare 50 mL of a sterile solution of 2% ultrapure agarose in 0.9% NaCl in ultrapure water. Autoclave the solution for 30 min at 121 °C.
2. Add 500 µL of sterile 2% ultrapure agarose solution in the center of a silicone mold containing 81 or 256 circular recesses.
3. After 40 min, unmold the ultrapure agarose from the silicone mold and place it in a well of a 12-well plastic plate.
4. Add 2 mL of DMEM LOW in the well with the micromolded agarose and incubate in an incubator at 37 °C in a 5% CO₂ atmosphere for at least 12 h before seeding the ASCs.

3. Biofabrication of ASC spheroids using the automated pipetting system

1. Check the following:
   1. Check whether laminar flow is on, and the airflow of the cabinet is working properly.
   2. Check whether the equipment is connected to the correct voltage.
   3. Check whether the tablet is connected to the equipment.
   4. Check whether the height of the cabinet protection glass is at the same height as the sensor marking of the equipment.
2. Press the On/Off button on the left side of the equipment and wait for the tablet and the equipment to start.
3. Position the tip boxes, the rack for centrifuge tubes, and the plate containing the micromolded agarose hydrogel in the workspace of the equipment.
4. At the tablet with the software open, first click on Labware Editor.
5. Set up a virtual worktable and choose the positions of the pipettes, tip boxes, the rack for the plastic tubes, and the plates.
   NOTE: The virtual worktable must represent the physical positions of the accessories in the equipment.
6. Click on the Switch to Produce button to include the parameters (see step 3.10) and commands that the equipment will carry out throughout the experiment. Wait for a toolbar and a Produce List to open in the software.
7. Initially, to indicate the commands, include the number of samples to be pipetted and drag it to the Produce List.
   NOTE: The number of samples are the number of plastic centrifuge tubes containing the ASC suspension to be seeded in the center of the micromolds.
8. After entering the number of samples, click on the Sample Transfer command button on the software to transfer the ASC suspension to the wells of the 12-well plate, containing the micromolds, and drag it to the Produce List.
9. Click on Properties to set up the start and end positions for the equipment to transfer the sample.
10. Wait for the software to return to the Work Table page. Click on the Options button to set up the parameters for automated seeding of the ASCs: i) Aspiration Speed of 15.4 s^-1; ii) Dispense Speed of 1 s^-1; iii) Blow Delay of 0 ms; iv) Blow Speed of 15.4 s^-1; v) Initial Stroke of 100%. Select Water as the Standard Liquid Type.
11. Set up the parameters to mix the ASCs to obtain a homogeneous suspension: i) Number of Cycles of 5; ii) Speed of 6.5 s^-1; iii) Volume of 300 µL.
12. After setting up the parameters, add the Time of 40 min to the Produce List.
    NOTE: This time is needed to wait for the ASC suspension to decant at the bottom of the micromold.
13. In the Produce List, include the Reagent Transfer option to transfer the spheroid culture medium to the corresponding wells of the plate.
14. Repeat steps 3.9-3.11 to set up the position and parameter configurations to transfer the spheroid culture medium.
15. Click on the button with a check symbol on the top bar of the software to ensure there is no programming error.
16. Click on the Play button (with a triangle symbol) on the top bar of the software to start the program.
17. Let the equipment start, as programmed, and wait for it to make a sound, indicating it has finished.
18. Collect the 12-well plate and incubate it in an incubator at 37 °C, 5% CO₂ for at least 18 h for the spheroids to be completely formed and compact.
19. Manually harvest the spheroids after 1, 3, and 7 days of culture for analysis. Manually flush the medium with a micropipette to liberate the spheroids from the micromolded non-adherent agarose hydrogel.
20. After 5 min, check under the microscope to determine whether the spheroids are completely released from the micromolded non-adherent agarose hydrogel.
21. Collect the spheroids manually using a micropipette and transfer them to a 15 mL centrifuge tube.
22. Wash the spheroids at least two times with 0.01 M PBS. Assess the viability and perform biomechanical analyses on the fresh spheroid samples. For morphology analysis (histology), incubate the spheroid samples in 4% paraformaldehyde in PBS for at least 18 h at 2-8 °C.

Results

The automatic pipette system can seed the ASC cell suspension into 12 wells of one 12-well plate in 15 min. Using the 81 micromolded non-adherent hydrogel will produce 972 spheroids at the end of the protocol. Using the 256 micromolded non-adherent hydrogel will produce 3,072 spheroids at the end of the protocol. ASC spheroids were analyzed for the homogeneity of their size and shape. ASC spheroids from micromolds with 81 recessions showed homogeneous diameter during the culture period in contrast to ASC spheroids from m...

Discussion

This paper presents the large-scale generation of ASC spheroids using an automated pipette system. The critical step of the protocol is to precisely set up the software to ensure the correct volume of cell suspension, speed, and distance for pipetting. The parameters described in the protocol were determined after a number of trials to optimize the dispensing of the ASC cell suspension into the wells of 12-well plates containing the micromolded, non-adherent hydrogels. The optimization was evaluated by measuring the diam...

Materials

Name	Company	Catalog Number	Comments
12-well plastic plate	Corning	3512
50 mL centrifuge tube	Corning	CLS430828
EpMotion 5070	Eppendorf	5070000282
epT.I.P.S. Motion	Eppendorf	30015231
ethylenediaminetetraacetic acid (EDTA)	Invitrogen	15576028
fetal bovine serum (FBS)	Gibco	10082147
Low Glucose Dulbecco's Modified Eagle Medium (DMEM LOW)	Gibco	31600034
MicroTissues 3D Petri Dish micro-mold spheroids - 16 x 16 array	Sigma	Z764000
MicroTissues 3D Petri Dish micro-mold spheroids - 9 x 9 array	Sigma	Z764019
phosphate saline buffer (PBS)	Sigma	806552
sodium chloride (NaCl)	Sigma	S8776
tissue culture flask	Corning	430720U
trypan	Lonza	17-942E
trypsin	Gibco	27250018
ultrapure agarose	Invitrogen	16500100