Co-culture Model Using Two Types of Adherent Cell Lines

Summary

The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

Abstract

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

Introduction

Despite extensive research on human pregnancy, the molecular mechanisms at the maternal-fetal interface during implantation and early pregnancy remain poorly understood¹. The human endometrium is mainly composed of two cell types: endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). Implantation progresses through three stages: apposition, attachment, and invasion, which lead to the development of a competent embryo and receptive endometrium². Considering the ethical constraints of in vivo studies on human subjects, as well as the difficulties in simulating the human condition in animals completely, the construction of in vitro human endometrium culture models has become an effective means of replicating the implantation and early pregnancy processes³. These models are valuable in investigating both normal and pathological pregnancies and provide a foundational platform for the preliminary testing and validation of therapeutic interventions in translational medicine.

Commercial chambers have been widely utilized in cellular biological research. These chambers offer valuable insights into cell migration and the crosstalk between different types of cells. However, commercial chambers are typically single-use and can be costly⁴.

A great number of in vitro human culture models consisting of endometrium and blastocyst, or blastocyst surrogates, have been developed to better understand the detailed process of implantation. These models, however, are still in their initial stage of application since the 3D structure is a highly complicated experimental setup, and certain specific differentiation media are expensive^5,6,7.

The use of affordable 3D-printing hardware and a relatively short manufacturing period makes it possible to fit structures to target different experimental purposes. 3D printing techniques help reduce time costs and enable the creation of complex, customized structures. This technology significantly accelerates prototype design and iteration, making it a valuable tool for researchers across various fields, allowing them to complete their work more efficiently^8,9,10.

Here, we present a feasible and economical experimental protocol for the construction of the 3D structure and its use as the cell culture system, which can simulate the interaction between endometrial stromal and epithelial cells for investigation of the endometrial receptivity during embryo implantation. This provides a customizable and low-cost alternative for commercial disposable material.

Protocol

NOTE: All reagents used in this protocol can be found in the Table of Materials. Unless otherwise specified, all media were pre-equilibrated to 37 °C before use.

1. 3D printing of the scaffold and model construction

NOTE: The steps here were performed according to the manual book of the commercial 3D-metal printing machine. The steps are briefly described below (Supplementary File 1).

1. Print bed leveling
   1. Tap the Menu icon on the dashboard and navigate to Utilities. Select Bed Level to initiate the leveling routine.
   2. Remove the print sheet from the print bed and select Done. The print bed will ascend to its maximum height in preparation for leveling. Once the progress bar reaches 100%, press Start.
   3. If the Metal X printer has not been leveled previously, select Reset. If it has, choose Skip and proceed directly to Step 1.1.5 (the 16-point scan).
   4. Use a 2.5 mm hex key to turn all three-bed leveling screws clockwise until they are tight. Then, loosen them by turning counterclockwise two full rotations to set them at a mid-point height. Press Done to continue.
   5. The printer will scan the bed at 16 designated points. Refrain from touching the bed or frame during this process. Once the progress reaches 100%, press Next.
2. Print sheet application
   1. Remove any residual metal or support debris. Toggle the Vacuum slider to On.
   2. Allow the bed to lower and heat up, then press Next. Place the print sheet over the vacuum grid and press Next.
   3. Position the sheet press over the print sheet, aligning it with the Z-rails at the rear of the chamber.
   4. Wait for the vacuum to engage and create a stable seal. Once the vacuum is engaged, press Done.
3. Metal filament loading
   1. Manually position the print head in the center of the printing area. Remove the print head cover by sliding it upward and off the mounting screws.
   2. Tap the Menu icon on the option board. Select Materials from the options.
   3. Choose Load Metal to start the loading routine. Choose Quick Load.
   4. Select the specific type of metal material to be loaded (e.g., Aluminum 6061-T6 | 3.3211 | 65028 | AlMg1SiCu).
   5. Put the spool on the holder and press Next. Close the door and allow the spools to warm up, then press Next.
   6. Insert the material into the front inlet of the print head (labeled M) until the extruder begins to draw it through.
4. Metal part printing
   1. In the Printing Settings panel, click Export Build. Save the file to a USB drive formatted to FAT32 and insert it into the printer.
   2. Select the Menu icon on the board. Navigate to Storage and select Print From Storage. Choose the part file to commence printing.
5. Metal part removing
   1. Carefully slide the print sheet and printed parts off the print bed. Gently detach the parts from the print sheet. The flexible sheet should peel away with ease.

2. Preparation for cell co-culture in the 3D model

NOTE: It is recommended that high-temperature resistant materials be used as consumables for 3D printing cell coverslip scaffold supports to facilitate autoclave sterilization before each use in cell culture experiments. Immortalized Human Endometrial Stromal Cells (HESC) and human endometrial epithelial cells (Ishikawa) were used in this study. The characterization of these cell lines has been previously reported^5,6.

1. Cell culture and passaging
   1. Prepare 50 mL of complemental medium for hESC by adding DMEM/F12 medium + 2 mM L-Glutamine + 10% charcoal-stripped Fetal Bovine Serum (FBS) + 1% Penicillin/Streptomycin Solution.
   2. Prepare 50 mL of complemental medium for Ishikawa by adding Dulbecco's Modified Eagle Medium + 10% FBS + 1% Penicillin/Streptomycin Solution.
   3. Cell thawing
      NOTE: The purchased cell line was shipped on dry ice in a frozen state. It should be stored in the vapor phase of liquid nitrogen or below -130 °C.
      1. Prepare all necessary sterilized equipment and dissociation enzyme, and warm the complemental medium to 37 °C.
      2. Thaw the cells rapidly in a 37 °C water bath with occasional gentle swaying. Transfer the cell suspension into a tube containing 5 mL of basal culture medium and centrifuge at 200 x g for 5 min.
      3. Carefully remove the supernatant. Resuspend the cell pellet in 5 mL of prepared complete culture medium and dispense it into two T25 culture flasks. Culture the cells in an incubator at 37 °C and 5% CO₂.
2. Preparation of cells with a coverslip
   1. Use an 18 mm square coverslip as the surface of the cell culture; make sure the coverslip is clean and sterile.
   2. Coat the coverslip in the bottom of a 12-well plate with 200 µL of extracellular matrix (ECM) at a concentration of 200-300 µg/mL. Keep the plate at 37 °C for 1 h.
   3. Remove the ECM from the plate. Inspect the cells for appropriate confluency, and make sure the density of both cell lines is around 70%-80%
   4. Remove the spent medium and wash the cells with 3 mL of phosphate-buffered saline (PBS) 2x.
   5. Add 2 mL of dissociation enzyme and culture the cells at 37 °C for 2 min to detach them.
   6. Neutralize with complete culture medium and create a single-cell suspension by pipetting up and down.
   7. Take 100 µL of the cell suspension and mix with trypan blue (dilution factor =2).
   8. Slide the coverslip over the chamber of the hemocytometer. Fill both side chambers with the cell suspension containing trypan blue.
   9. Count the number of cells in squares on both sides under a 20x microscope and calculate the density of cells.
   10. Transfer the cells to the prepared plate with fresh medium and incubate at 37 °C with 5% CO₂. Seed hESC into the well at 1 x 10⁵ cells/mL for 24 h.
   11. For the Ishikawa cell line, seed the cell into the well without a coverslip the next day.

3. Assembly of the co-culture model

1. Soak the scaffolds with ethyl alcohol and double distilled water (ddw) thoroughly for 2 days. Sterilize scaffolds with an autoclave at 121 °C and dry them.
2. Add about 2 mL of complemental medium for the Ishikawa cell line to the well culturing Ishikawa, ensuring the liquid level covers the coverslip.
3. Transfer the coverslip cover with hESC onto the scaffold and keep the side with the cell down. Plug the scaffold into the well-culturing Ishikawa. For the second set, invert this arrangement by transferring the coverslip cover with Ishikawa onto the scaffold and plugging the scaffold into the well-culturing hESC. Use the cells without the scaffold setup for independently growing cell growth analysis.
4. Incubate the co-culture system at 37 °C, 5% CO₂. The co-culture period can vary depending on the experimental design but typically ranges from 24-72 h.

4. Image acquisition

1. Carefully remove the culture medium and rinse cells gently with sterile phosphate-buffered saline (PBS).
2. Remove coverslips with attached cells from culture dishes using fine forceps or tweezers. Optionally, place coverslips in a new dish with PBS to prevent drying during transfer.
3. Take a clean microscope slide and place a drop of PBS. Gently transfer the coverslip containing cells onto the drop of mounting medium, ensuring cells face down onto the slide.
4. Place the prepared slide onto the stage of the inverted microscope. Select the appropriate objective lens (e.g., 10x, 20x) and adjust focus using coarse and fine focus knobs.
5. Set microscope parameters such as illumination intensity and camera settings. Using the microscope software, capture images of the cells at desired magnifications and fields of view. Adjust exposure time and other imaging settings to optimize image quality.

5. Image processing

1. Click Open to open the image, click Process > Filters > Enhancement in order > Choose Local Equalization, and click Apply.
2. Click Edit > Convert To > Gray Scale 8. Click Enhance, choose Apply Contrast.
3. Click Count and measure subjects. Click Manual, select a range, and adjust the histogram to make sure that all the cells are covered.
4. Click Apply Mask, close the current window, Click Automatic Bright Objects > Measure, Select Measurement, and choose Aera, Dendric, Density, and Size.
5. Click Automatic Bright Objects, then click Count. Click Export Data to export the measurement data to a spreadsheet.

6. Cell harvesting

1. Remove coverslips from the microscope slide and transfer them to a clean and dry well.
2. Harvest the Ishikawa cell with RNA isolation reagent for transcriptomic or Ripa lysis reagent for proteomic research.
   NOTE: For hESC, the coverslip-culture method was more effective for image capture after immune staining. Alternatively, the hESC could be harvested with RNA isolation reagent or Ripa lysis reagent. Cell lines cultured on the coverslip or the surface of the plate could be exchanged; however, we recommend that the cells prepared for imaging analysis be seeded on the coverslip.

Results

Figure 1 shows the homemade scaffold for cell slides used in this process, which comprises an upper support ring tailored for attachment to standard 12-well cell culture plates, complemented by a basal cell slide holder featuring four L-shaped rod-like structures.

According to Figure 2, the density of both cell types in the co-culture condition (Figure 2A,B) remained low after 72 h of co-culture; however, the density of both cell types cultured independently grew significantly (Figure 2C,D). Moreover, the length of co-cultured hESC was shorter compared with independently cultured hESC (Figure 2E-G).

The profile of Ishikawa cells and hESC cells indicates the existence of interactions between the two types of cells. At the same time, the system enables us to continue to perform follow-up experiments on the separate cell lines, including immunochemistry staining and western blot.

Figure 1: 3D-printed coverslip scaffold utilized in the experimental protocol. (A) Scaffold in mono-form. (B) Scaffold in the binary form. (C) Illustration demonstrating the placement of coverslip on the scaffold. (D) Illustration demonstrating the placement of scaffold in a standard 12-well plate. Please click here to view a larger version of this figure.

Figure 2: Profile of hESC and Ishikawa cell lines treated for 3 days with different conditions. (A) The Ishikawa seed on the coverslip co-cultured with hESC by scaffold (Bar=200 µm). (B) The hESC seed on the coverslip co-cultured with Ishikawa using a scaffold. (C) The Ishikawa cells were cultured in a complemental medium with 10% FBS. (D) The hESC cells were cultured in a complemental medium with 10% FBS. (E) The cell density of hESCs under co-culture conditions is significantly lower than that under independent-culture conditions. (F) The cell density of ISHIKAWA cells under co-culture conditions is significantly lower than that under independent culture conditions. (G) The length of hESC under co-culture conditions is significantly shorter. N=3; ***, p<0.05, the error bar shows standard error. Please click here to view a larger version of this figure.

Supplementary File 1: 3D printing coding file. Please click here to download this File.

Discussion

A simplified and cost-effective protocol is described for the indirect co-culture of endometrial stromal and epithelial cells. This method utilizes a homemade scaffold for cell slides, which comprises an upper support ring tailored for attachment to standard 12-well cell culture plates, complemented by a basal cell slide holder featuring four L-shaped rod-like structures. This setup facilitates the separation of endometrial stromal and epithelial cells while allowing for their interaction through the exchange of signaling molecules in the culture medium. One of the key advantages of this approach is the substantial reduction in both time and expenditure compared to commercially available co-culture systems¹¹. The homemade scaffold is constructed from readily available materials, significantly lowering the cost of the setup. Additionally, its design is straightforward, enabling researchers to assemble and disassemble the scaffold with ease, thus saving time and effort in the laboratory. Moreover, the homemade scaffold can be cleaned and sterilized for multiple uses, making it a sustainable and economical option for ongoing research. This feature is particularly beneficial for long-term studies, where consistent and reliable results are crucial.

Endometrial stromal cells and epithelial cells play pivotal roles in facilitating successful embryo implantation. Under physiological conditions, endometrial epithelial cells and stromal cells in the endometrium do not typically have direct contact.Research on human endometrial stromal cell decidualization has proved that epithelial cells influence the decidual process by paracrine. The close proximity of epithelial and stromal cells allows for effective communication through paracrine signaling¹². This interaction is vital for coordinating the cyclical changes in the endometrium, including the preparation for implantation and subsequent tissue shedding if implantation does not occur¹¹. Both epithelial and stromal cells express receptors for estrogen and progesterone, which regulate their proliferation, differentiation, and secretory activities. The epithelial-stromal crosstalk ensures that these responses are well-coordinated¹³. Thus, the complex crosstalk between these cells remains a critical area of investigation, as it is essential to understand the intricate processes that underlie endometrial receptivity and the implantation of the embryo¹⁴. Despite significant advances in reproductive biology, the detailed mechanisms of communication between these cell types during implantation are still not fully elucidated¹⁵.

The scaffold provides a convenient way to achieve the co-culture of two kinds of adherent cells. However, in the process of long-term co-culture, the coverslip cells continue to grow and renew over time, and the dead cells will fall and come into contact with the lower-layer cells, which may impact the lower-layer cells. In addition, the cells seed on the coverslip are placed face down on the scaffold. Whether gravity has any effect on the state of the cells is unknown. In this research, two cell lines, hESC and Ishikawa, were used for demonstration. The scaffold can be widely used in the co-culture of two different kinds of adherent cell lines. In addition, the scaffold can also be used as a platform for other compounds or medicine release media and as a mounting platform for various devices such as luminescence, heating, and electrode devices.

Here, we present a reliable and robust in vitro cell culture system utilizing the specific scaffold, which is a useful and convenient tool to study the molecular mechanism of embryo-maternal uterine cell interactions further. In addition, we have characterized the feasibility of the established scaffold as a model for blastocyst/trophoblast studies. This protocol allows the construction of suitable scaffolds by 3D printing and their function as the 3D structure for the migration or invasion platforms in small volumes (12-well plates), where different factors can be evaluated using imaging quantification systems.

The use of cell lines allows the standardization of the model to compare different conditions, something that is not applicable using fresh primary villi tissues. However, any of the cell line components can be replaced by primary culture for validation. By incorporating the cells and tissues derived from patients of interest, the proposed model may facilitate the demonstration and modeling of different diseases that have been known to contribute to infertility and pregnancy failure.

Critical steps
Firstly, the density of Ishikawa cells for co-culture should be limited, approximately 60%-70% confluency. Secondly, the coverslip cover with cells should be kept down. Thirdly, the concentration of ECM should be less than 3 mg/mL to avoid forming a gel. Fourthly, the liquid level of the medium in the co-culture system should cover the coverslip.

Limitations
The model mentioned above comprises the endometrial epithelial cells and the stromal cells; one limitation of the study was the use of adenocarcinoma Ishikawa cells as epithelial cell surrogates instead of primary cells. Integrating primary epithelial and stromal cells from the same patient would replicate the physiological conditions of the human endometrium. For some experimental applications, a possible limitation concerns the lack of other cell types, such as endothelial cells and immune cells, and there is no characterization of the glandular structure in this model to assess the interactive impact of the endometrial glands on other endometrial cell types. However, under physiological conditions, the epithelial cells reveal polarity, and this character hardly simulates in vitro conditions without a uterine luminal environment. The endometrial stromal cells are under the decidual process in the menstrual cycle^16,17. However, the molecular mechanism of decidualization remains unclear. Several factors, including immunocytes and endothelial cells, could play a role in decidualization. This model provides a limited way of examining the indirect crosstalk between stromal and epithelial cells during the decidual process. Furthermore, the levels and roles of the biological markers during decidualization are still unclear, which warrants further exploration

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x Hanks′ Balanced Salt solution	Solarbio	H1046	1/10
12-well Clear TC-treated Plates	Corning	3513	-
25 cm² Cell Culture Flask	Corning	430639	-
Aluminum	Markforged	6061-T6	-
DMEM/F12	Sigma-aldrich	D2906	-
Dulbecco’s Modified Eagle Medium	Gibco	C11995500BT
FBS	Gibco	10099141C	1/10
Fetal bovine serum	Gibco	10099141C
ITS Premix	Biocoat	354350	1/100
Matrigel Matrix	Corning	354248	ECM
Metal X	Markforged	M F-PR-5000	-
Penicillin-Streptomycin	Gibco	15140122	1/100
Round Coverslip	Biosharp	BS-18-RC	-
TrypLE Select (10X)	Gibco	A1217701	dissociation enzyme