Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Summary

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Abstract

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 µm. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and >95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Introduction

Tissue spheroids are 3D micro-tissues formed by cell suspensions that undergo self-assembly without external forces¹. These spheroids have been widely used in biofabrication protocols due to their resemblance with key features of the human physiological system^2,3. Tissue spheroids provide more similar metabolism, cytoskeleton dynamics, cell viability, and metabolic and secretion activity than traditional monolayer cell culture¹. Due to their fusion capability, they can also be used as building blocks (e.g., bioprinting protocols) to form complex tissue-engineered constructs with enhanced biological relevance^4,5.

Due to their biological relevance, tissue spheroids have been used as a biotechnological tool for protocols ranging across tissue engineering, drug development, disease modeling, and nanotoxicological assessment, reducing time, space costs, and animal testing^3,6,7,8. Nonetheless, to fully explore and leverage the potential of tissue spheroids, reliable and reproducible methods aiming at their large-scale production are highly necessary, and these remain an ongoing challenge.

Several methodologies produce spheroids, such as hanging drop, coated u-shaped bottom wells, microfluidics, and using a polymeric matrix^9,10. Although these methodologies paved the way within the spheroid production market, they are still complex, time-consuming, labor-intensive, or expensive¹⁰.

The present protocol reports the large-scale production of homogeneous tissue spheroids using a low-cost and time-effective methodology. We have developed a 3D printed stamp-like device to generate up to 4,716 spheroids per 6-well plate. Moreover, the stamp-like device can be tailored to produce more spheroids per well, suitable for different cell culture plates. It is easily sterilizable and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to translate their use to the clinics, contributing to multiple areas of industry such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Protocol

The L929 cell line, mouse fibroblasts, was used for the present study. The stamp-like 3D printed biodevice was obtained from a commercial source (see Table of Materials). Good cell culture practice and sterile techniques were followed throughout the protocol. The fabricated device was sterilized by wiping it with 70% alcohol and exposing it to UV light for 15 min. The cell culture media and solutions were warmed to 37 °C before contacting with the cells or tissue spheroids. A schematic representation of the protocol is shown in Figure 1.

1. Preparation of non-adherent molds from the stamp-like device

1. Prepare 2% (w/v) agarose gel following the steps below.
   1. Dilute the agarose powder in phosphate-buffered saline (1x PBS) and homogenize the resulting suspension with circular movements.
      NOTE: This solution can be placed in a glass bottle. In this step, the agarose solution will not be translucent.
   2. Place the glass bottle into the microwave and set it for 30 s. Every 5 s, stop the microwave, remove the glass bottle, and manually homogenize the solution with circular movements. The heating process needs to be performed until the solution reaches a liquid-limpid state.
      NOTE: A hot plate can also be used as a microwave alternative. After the heating process, the solution must be translucent/limpid.
   3. Add 1 mL of the agarose solution to each well of a 6-well plate planned for the experiment.
   4. Wait for ~15 min or until the agarose solidifies.
      NOTE: A cooling plate can be used to decrease the solidification time.
   5. Add 1-2 mL of the agarose solution and gently insert the device above the liquid agarose.
      NOTE: The placement of the device must be done carefully to prevent air bubbles in the agarose-device interface.
   6. Wait for ~30 min or until the agarose solidifies.
      NOTE: A cooling plate can be used to decrease the solidification time.
   7. Gently remove the device from the agarose.
      NOTE: The removal is critical. One should remove it carefully to maintain the agarose features intact; otherwise, the agarose can be disrupted.
   8. Add 2 mL of DMEM media, wait for 10 min, discard the media, and replace it with fresh DMEM. Repeat this three times to wash the well properly.
   9. Add 2 mL of DMEM and place the 6-well plate in an incubator (at 37 °C in 5% CO₂ and 80% humidity) until the cell seeding (Figure 2A-F, Supplementary Video 1).

2. Generation of tissue spheroids

NOTE: Different cell lineages have different adhesion properties. Hence, using this methodology, some types of cells may not form the tissue spheroids properly.

1. Grow the cells following traditional monolayer culture (i.e., grow the cells in cell culture flasks using DMEM with low glucose supplemented with 10% fetal bovine serum (FBS), 100 µg/mL penicillin, and 100 µg/mL streptomycin) (see Table of Materials). Maintain the cells at 37 °C in a 5% CO₂ incubator, and monitor until they reach 80% of confluence.
2. After reaching the desirable confluence, wash the cells with 1x PBS.
   NOTE: It is recommended to use 5 mL for 25 cm² flasks , 10 mL for 75 cm² flasks, and 15 mL for 150 cm² flasks.
3. Add the dissociation enzyme and incubate the cells for 2-5 min at 37 °C in 5% CO₂ and 80% humidity.
   NOTE: The present study used 0.125% trypsin with 0.78 mM ethylenediamine tetraacetic acid (EDTA) as the dissociation enzyme (see Table of Materials).
4. Observe the detachment of the cells from the cell culture flasks and add a growth medium supplemented with FBS to neutralize the cell dissociation enzyme.
   NOTE: For the present study, DMEM with low glucose (see Table of Materials) was used supplemented with 10% FBS.
5. Centrifuge the cell suspension at 400 x g for 5 min at room temperature. Then, count the cells manually.
6. Take 50 x 10⁵ cells per tube and add 5 mL of 1x PBS.
   NOTE: The number of cells seeded influences the final tissue spheroid diameter. Hence, one can increase the cell number to generate tissue spheroids with larger diameters.
7. Centrifuge the cell suspension at 400 x g for 5 min at room temperature.
8. Remove the supernatant using a pipette, add 1 mL of the cell culture medium, and homogenize the solution.
   NOTE: In the present study, a complete culture medium of DMEM with low glucose, supplemented with 10% FBS, 100 µg/mL penicillin, and 100 µg/mL streptomycin, was used.
9. Remove 2 mL of medium from the 6-well plate (step 1.1.9) and add 1 mL of the cell suspension to the center of the agarose mold formed by the 3D-printed biodevice (step 1.1.7). Wait until the cells sediment in the micro resections (~20-30 min) and carefully add 1 mL of cell culture medium into the well.
   NOTE: One needs to be extra careful in this step. It is recommended to gently add the medium, place the pipette tip close to the well wall, and dispense in small amounts.
10. Place the 6-well plate in the incubator (at 37 °C in 5% CO₂ and 80% humidity) for the tissue spheroids to form (approximately 24-48 h, depending on the cell type) (Figure 3).
    NOTE: Different cell types (e.g., cancer cells, primary cells) have different self-assembly kinetics¹¹.

Results

Generation of homogeneous micro resections using the 3D printed stamp-like device
The 3D printed stamp-like device was successfully manufactured by the stereolithography method¹² using a photocurable resin (Figure 2A). The final device was composed of cylindrical micropins with a height of 1.3 mm and a width of 650 µm (Figure 2A). Its use as a master mold to produce non-adherent micro resections was achieved by...

Discussion

The present protocol describes a simple, fast, and inexpensive method for the large-scale production of tissue spheroids. A stamp-like 3D printed device was used as a master mold, which generated up to 4,716 spheroids per 6-well plate. It has been shown that cells cultivated as spheroids have more realistic biological responses that closely resemble the in vivo environment¹. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more pred...

Materials

Name	Company	Catalog Number	Comments
6 well plate	Merck	CLS3516
Agarose	Promega	V3121
Biodevice	Bioedtech
Biological Safety Cabinet	ThermoFisher	51029701
Centrifugue	ThermoFisher	75004031
Corning 50 mL centrifuge tubes	Merck	CLS430829-500EA
Corning cell culture flasks surface area 75 cm2	Merck	CLS430641
Draft Resin	FormLabs	FLDRBL01
Dulbecco′s Modified Eagle′s Medium - low glucose	Merck	D6046
Fetal Bovine Serum (FBS)	ThermoFisher	16000044
Form 2	FormLabs
Incubator	ThermoFisher	51033782
L929 cell lines	Stablished in the lab
Penicillin and Streptomycin (PS)	ThermoFisher	15140122
Phosphate-Buffered Saline (PBS)	Merck	806552
Trypsin with EDTA	Merck	T3924