Cell Line-Derived Xenograft Using Xeno-Free VitroGel System in Comparison to Animal-based Extracellular Matrix

Materials and Methods

Overview of cell lines and hydrogel systems

The tumor development study was conducted in 60 athymic nude CDX mice models. The age of the mice chosen ranged from 6–8 weeks at the time of inoculation. All mice for this experiment were supplied by Charles River Laboratories. The VitroGel Hydrogel Matrix (TheWell Biosciences; catalog number: VHM01) and AB-ECM acted as the delivery system for the injection of specific tumor cell lines. Six cell lines, PC3, HCT-116, Calu-6, HCC1806, HeLa, and PANC1, were inoculated into the mice after mixing with either VitroGel or AB-ECM in a paired fashion, with five mice per treatment being studied (Table 1). These cell lines spanned several human tissue sources, including prostate, colorectal, lung, ovary, pancreas, and breast (Table 1). Mice and their tumors were observed over a period expressed in days for the treatment group with Hydrogel and AB-ECM to compare the time needed for each to reach the predetermined tumor size within a time frame of 2–10 weeks.

 Cell culture media and handling of VitroGel versus AB-ECM

Cells were sub-cultured in specific media (Table 1), and those growing in the exponential phase were harvested. Cells were then resuspended in PBS without the addition of more supplements. After one round of filtration and cell counting, the cell suspension was followed by another round of centrifugation at 335 x g RCF, after which PBS was used to resuspend the cells to get 2X of desired cell concentrations.

Following this step, the protocols used for VitroGel versus AB-ECM were initiated with slight variations. For the methodology using VitroGel, the product was first brought to room temperature. The cell suspension (2X) was mixed with VitroGel at a ratio of 1:1 (v/v). After being transferred to the syringe, this mixture was left at room temperature for 10–15 minutes, during which time crosslinking starts to stabilize before injection. The injection volume was either 100 μL or 200 μL, depending on the cell type, to obtain the required cell density (1 x 10⁶, 2 x 10⁶, or 10 x 10^6, depending on cell type; see Table 1). The injection was performed subcutaneously near the dorsal thigh region of the mouse.

For AB-ECM, the solution was thawed and kept at 4 °C throughout the protocol. The cell suspension (2X) was then mixed with AB-ECM at a 1:1 ratio (v/v). Again, the loading volume was 100 μL or 200 μL to obtain the desired number of cells; the number of cells for each cell type was equalized for the paired VitroGel and AB-ECM experiments. Following each round of inoculation, the syringe was put back on ice to prevent suspension solidification and needle clogging. Details of protocol components can be found in Table 1 below.

Table 1. Details of media, injection number, volume, and strain remained similar for use with both VitroGel and AB-ECM.

Cell line	Culture media	Cell injection #	Injection volume	Mouse strain
PC3	RPMI-1640, 10% FBS	2 x 106	100 μL	Nude mouse
HeLa	EMEM, 10% FBS	1 x 106	100 μL	Nude mouse
HCT-116	McCoy’s 5A, 10% FBS	10 x 106	200 μL	Nude mouse
HCC1806	RPMI-1640, 10% FBS	10 x 106	100 μL	Nude mouse
Calu-6	EMEM, 10% FBS	10 x 106	100 μL	Nude mouse
PANC1	DMEM, 10% FBS	10 x 106	200 μL	Nude mouse

Tumor Tracking

Tumor size was measured two times per week using a caliper. Volumes were expressed in cubic millimeters using the formula: V (mm³) = (a (length) × b (width)²)/2, where a and b are the long and short diameters of the tumor, respectively. Averages and standard deviations among the (max = five; individuals were removed from the study upon >20% body weight loss, excessive stress, or death) mouse samples at each time point were calculated using Microsoft Excel. Experiments were continued until a predetermined tumor size (e.g., 1000 mm³) was reached. Animals were kept at room temperature (22 °C ± 3 °C) in 50% (±20%) humidity with a 12 h/12 h day/light cycle throughout the experiment. Appropriate IACUC approval was obtained prior to the initiation of the experiments, and ethical treatment of all animals was followed.

Results

The observations were made over 2–10 weeks, depending on the tumor growth rate. A solid tumor was found to be forming successfully for all of the six solid tumor-derived cell lines when using either VitroGel or AB-ECM; tumor types and morphologies were analogous. The average growth rate was nearly similar in both AB-ECM and VitroGel for all cell types (Table 2; Figure 2). For all the six cell types, it was found that the time required to achieve the predetermined tumor size (Table 2) was the same in AB-ECM and VitroGel Hydrogel. Mice that showed a tumor growth size of greater than 2000 mm³ or those for which tumors exceeded 2 cm in any one dimension were neglected. Tumors grafted using VitroGel also displayed a lower standard deviation in most cases, implicating VitroGel as a reliable system that comes with less variability and more batch-to-batch consistency. These characteristics demonstrated the efficiency of VitroGel Hydrogel as a xeno-free substitute to AB-ECM, allowing it to be a promising alternative with the added ease of application. With superior properties like long-term injectable status, bulk sample preparation, and easy-to-use room temperature operation, VitroGel hence acts as a powerful tool for in vivo studies.

Table 2. Tumor growth endpoint times and tumor sizes for all six cell types in VitroGel.

Cell type	Tumor growth endpoint	Final tumor volume (predetermined)
PC3	8 weeks	1000 mm3
HeLa	7–8 weeks	400 mm3
HCT-116	2–3 weeks	2000 mm3
HCC1806	2–3 weeks	2000 mm3
Calu-6	4–5 weeks	2000 mm3
PANC1	8–9 weeks	400 mm3

Discussion

The tumor growth curves, as seen above in Figure 2, indicate that there was successful solid tumor formation for all of the six cell types tested. The time required to reach a predetermined tumor size was comparable in both the AB-ECM and VitroGel, indicating the efficacy of VitroGel as a promising alternative to animal-based extracellular matrix for in vivo applications. Besides the six cell types above, VitroGel Hydrogel Matrix also supports many xenograft models and cell types: Breast (Hs578T, MDA-MB-231), Leukemia (CCRF-CEM, Reh), Lung (A549, H2170), Melanoma (A375), Pancreas (ASPC-1), Oral Cavity (HSC-2), Fibrosarcoma (HT1080), Renal (786-O), Tongue (CAL-27) and more.

Apart from the tumor growth success, coupled with no toxicity signs post-injection, VitroGel serves as a system that encompasses many practical benefits for cancer scientists. It helps encounter the challenge of maintaining a constant cold temperature of 4 °C during the pre-injection process, making its usage more flexible when ice is unavailable or inconvenient. A homogeneous cell suspension before injection, followed by a smooth injection administration, ensures reproducibility. With batch-to-batch consistency when compared to its AB-ECM counterpart, VitroGel is a reliable system for long-term analyses.

The system is intended to not only act as an alternative to AB-ECM but also to provide scientists the ability to control the supplements and growth factors they wish to add. This synthetic hydrogel system allows scientists to manipulate the key biophysical and biological properties such as mechanical strength, functional ligands, and degradability of the microenvironment.  Hence it stands for a powerful biocompatible tool that lets one create many unique assays that native ECM can not do. Besides xenograft, the injectable VitroGel system is also excellent for other in vivo applications such as cell therapy, drug delivery, and wound healing.^4-8

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