Frequently Asked Questions on 3D Cell Culture Matrices 

NexaGel^® is a xeno-free, versatile hydrogel system designed for 3D cell culture, 
tissue engineering, and regenerative medicine applications.

NexaGel^® is composed of synthetic polymers that mimic the extracellular matrix, providing a supportive environment for cell growth and differentiation.

The pore size of NexaGel^® is optimized for nutrient exchange and cell migration,
supporting various cell types and applications.

No, NexaGel^® does not contain natural ECM proteins, allowing researchers to customize the matrix with specific proteins or growth factors.

The hydrogel formation starts when NexaGel^® is mixed with the cell culture medium. The hydrogel molecules will interact with the ionic molecules, such as Ca2+ or Na+, from the cell culture medium to induce a matrix structure (hydrogel). For 3D culture operation, the hydrogel formation is in two stages: a) soft hydrogel formation and b) hydrogel stabilization.

Soft Hydrogel Formation: The process is slow when a small amount of cell culture medium is used. At this stage, the hydrogel is soft and possesses a shear-thinning and rapid-recovering mechanical property, which makes it injectable for in vivo applications. The slow hydrogel-forming process and the injectable property of the soft hydrogel create a time frame for easy hydrogel transfer from the mixing tube to the cell culture plate.

Hydrogel Stabilization: After soft hydrogel formation, adding cell culture medium on top of the hydrogel would allow more ionic molecules to penetrate the hydrogel matrix and further saturate the hydrogel cross-linking. A solid hydrogel would form during this process.

Every NexaGel^® hydrogel undergoes testing with a dynamic rheometer to assess its viscoelastic properties.

For NexaGel^® hydrogels with high concentration, the elastic modulus (G’) can be modified from 10 to 4,000 Pa by adjusting the hydrogel concentration using the NexaGel^® Dilution Solution. You can convert the elastic modulus provided on our website to stiffness using the table below (1 Pa of G’ corresponds to approximately 3 Pa of stiffness).

Before gelation, the viscosity of NexaGel^® solution is less than 0.1 Pa·s. Additionally, increasing the temperature, such as to 37°C, can further decrease the viscosity of the hydrogel solution.

The stiffness of the ready-to-use hydrogels varies and can range from 100 - 1000 Pa.

We measure the elastic modulus (G’) of NexaGel^® using a dynamic rheometer. The G’ is influenced by the hydrogel dilution and the cell culture medium applied. For non-diluted NexaGel^® High Concentration, the G’ is approximately 4,000 Pa.

NexaGel^® Dilution Solution Type 1 includes sucrose to ensure optimal osmolarity, while Type 2 is free of sucrose. For the majority of cell lines, NexaGel^® Dilution Solution Type 1 is advised. NexaGel^® Dilution Solution Type 2 is designed for researchers working with cells that are sensitive to sugar.

Conjugated

Yes, NexaGel^® facilitates the diffusion of molecules of various sizes, ranging from small molecules to large proteins like IgG. Drug compounds, antibodies, and staining dyes can be incorporated into the cover medium to permeate the hydrogel. NexaGel^® can also be combined with molecules for controlled release experiments.

Ready-To-Use Hydrogels: For many users, the "Ready-To-Use" hydrogels are preferred for various applications. You simply need to add your cells, cover with medium, and incubate. These hydrogel versions have been formulated and optimized for different cell types and applications.

NexaGel^® Hydrogel Matrix (Material number: NGH01): This is the recommended hydrogel for many users, particularly for 3D cell culture and 2D coating. It is a formulation of multi-functional ligands and concentrations to support various cell types for different applications. It is ready-to-use; you only need to add your cells, cover with medium, and incubate.

NexaGel^® STEM (NGH02): This is specifically designed for the 3D culture and expansion of human pluripotent stem cells (hPSCs). It is optimized to maintain stemness, support high cell viability, and enable the formation of uniform 3D spheroids. The hydrogel is ready-to-use and suitable for scale-up, organoid differentiation workflows, or re-establishing 2D cultures after expansion.

NexaGel^® MSC (NGH03): This is tailored for the 3D culture of mesenchymal stromal/stem cells (MSCs), supporting cell survival, proliferation, and functional phenotype. Its formulation provides an ECM-mimicking environment suitable for MSC expansion, differentiation studies, and regenerative medicine research. The hydrogel is ready-to-use, requiring only cell addition, medium overlay, and incubation.

NexaGel^® ORGANOID Discovery Kit (NGH04-K): The NexaGel^® ORGANOID Discovery Kit contains four distinct organoid hydrogel formulations (ORGANOID 1–4) with different mechanical strengths and bio-functional properties. It is designed to help researchers identify the optimal matrix for their specific organoid type, including patient-derived and PSC-derived organoids, co-cultures, and cancer models. The kit supports both 3D encapsulation and dome cultures and is ideal for method development and screening.

NexaGel^® HEK293 (NGH05): This is optimized for the 3D culture and suspension growth of HEK293 cells, commonly used in protein production and transfection workflows. The hydrogel supports high cell viability, uniform cell distribution, and scalability, making it suitable for recombinant protein expression, viral vector production, and functional assays. It is ready-to-use and integrates easily into standard cell culture workflows.

High Concentration (Tunable) Hydrogels: "Building Blocks for Tissue Engineers"
For more advanced users, high-concentration hydrogels provide scientists with full control to manipulate the biophysical and biological properties of the cell culture environment. All high-concentration hydrogels come with NexaGel^® Dilution Solution to adjust the final hydrogel strength from 10 to 4,000 Pa.

NexaGel^® 3D High Concentration Kit (Material number: NGH001): A pure, unmodified hydrogel that allows maximum flexibility to manipulate the 3D cell culture environment for different needs. The unmodified hydrogel matrix structure is suitable for cell spheroid formation, suspension cells, or cells requiring low cell-matrix interactions.

NexaGel^® RGD High Concentration Kit (Material number: NGH003): Modified with a high concentration of RGD cell adhesive peptides, promoting cell attachment and cell-matrix interactions during 3D cell culture. It can achieve high levels of integrin-binding activities to promote intercellular networks even after hydrogel dilution.

NexaGel^® COL High Concentration Kit (Material number: NGH009): Modified with a collagen-mimetic (GFOGER) peptide that specifically binds the integrin α2β1. This promotes many bioactivities, such as osteoblastic differentiation in vitro and enhancing osteoblastic activity in vivo.

NexaGel^® MMP High Concentration Kit (Material number: NGH010): Modified with Matrix Metalloproteinases (MMP) for a biodegradable matrix. We can customize our hydrogels with different functional ligands.

NexaGel^® IKVAV High Concentration Kit (Material number: NGH007): Modified with laminin-derived functional peptide, which is actively involved in various biological activities such as neuronal progenitor cell differentiation, promoting cell adhesion, neurite outgrowth, angiogenesis, and tumor growth.

NexaGel^® YIGSR High Concentration Kit (Material number: NGH008): Modified with a laminin-derived functional peptide that involves endothelial cell adhesion, cell proliferation, and motility/migration.

We have conducted tests on cell growth within the NexaGel^® hydrogel system for over eight weeks. Depending on the cell type and application, the 3D cell culture can extend even further. As the number and size of cells grow, researchers may need to change the cover media more frequently.

Yes, researchers can easily and quickly harvest cells using our enzyme-free, ready-to-use NexaGel^® Cell Recovery Solution in just 20 minutes. NexaGel^® Cell Recovery Solution is stable at room temperature, has a neutral pH, and operates at 37°C. This cell harvesting solution maintains high cell viability during the recovery process. Harvested cells can be further subcultured for both 2D and 3D applications.

It is a non-enzymatic cell harvesting solution that recovers cells/organoids from hydrogel or an animal-based ECM within 15 minutes, yielding high-quality cells/organoids for downstream analysis or expansion.

Our NexaGel^® Organoid Recovery Solution (NGR04-100, NGR04-500) is not highly effective for collagen. Some stem cells, like MSCs, when cultured in hydrogel for an extended period, produce their own ECM, primarily collagen, which further crosslinks the hydrogel. In such situations, an enzyme or proteinase needs to be added.

Yes, NexaGel^® is biocompatible and safe for animal studies.

Yes, NexaGel^® can be injected either before or after soft hydrogel formation for in vivo studies.

Before hydrogel formation, the NexaGel^® solution can be directly injected into the animal, where it will transform into a hydrogel upon encountering the ionic compounds of the physiological environment. After soft hydrogel formation, the NexaGel^® hydrogel possesses enhanced injectable properties. By mixing the hydrogel solution with cell culture media or PBS at an appropriate ratio, the final hydrogel becomes suitable for injection in in vivo studies. This method allows cells or other chemical compounds to be incorporated into the hydrogel prior to injection.

The gel may develop a sponge-like structure. If a very diluted NexaGel^® hydrogel is used to coat the plate, it will form a thin layer once dried.

Yes, NexaGel^® can be used for cryopreservation, aiding in the promotion of cell viability.

Yes, DMSO can be used with NexaGel^®. We recommend using no more than 50% of the volume as the maximum amount of DMSO.

NexaGel^® is a xeno-free system, and no autofluorescence has been detected in our various imaging tests. We recommend that researchers conduct a control experiment to assess background fluorescence.

In most cases, we do not recommend preparing pre-diluted NexaGel^®. Once the NexaGel^® solution is mixed with the dilution solution, the hydrogel will begin to slowly crosslink. Although this initial gelation keeps the gel injectable, once crosslinking starts, the functional ligands in the hydrogel will gradually be neutralized by the salts in the medium over time.

Ideally, we recommend preparing the hydrogel fresh for each use. The non-diluted NexaGel^® solution can be stored at 4°C for several months. There is no need to pre-dilute or pre-mix the hydrogel before use.

Prepare the cell suspension in a serum-free medium to maintain cell viability and functionality. Serum can be added if required for specific applications

Hydrogel formation time can be adjusted by varying the temperature and mixing ratio of the hydrogel and cell suspension.

Transfer the sample quickly to prevent premature gelation and ensure even distribution in the culture plate.

No, the hydrogel matrix supports the cells, preventing them from sinking and ensuring uniform distribution.

Yes, serum-free medium works with NexaGel^®.

Yes, when diluted, the viscosity will be reduced.

Yes, extracellular matrix proteins or other molecular compounds can be incorporated into the NexaGel^® system. Before hydrogel formation, add the desired proteins or compounds to the cell culture medium and then mix directly with the NexaGel^® hydrogel solution. Please note that the gelation time and final stiffness of the hydrogel may be affected by the salts present in the proteins or chemical compounds. If you have any questions or concerns about adding additional compounds to the NexaGel^® system, please contact us.

Yes, there are visual indicators to help confirm the formation of NexaGel^® hydrogel. Typically, an initial soft gel forms within approximately 20 minutes at room temperature. Once the cover medium is added, a distinct separation between the NexaGel^® hydrogel and the cover medium becomes visible. This indicates that the gel is ready for incubation at 37°C for long-term cell culture.

Yes, that is doable. 

Bubble formation is often due to the increased viscosity that occurs when NexaGel^® is mixed with cell culture medium. To reduce bubbles during preparation, consider the following tips:

Warm the NexaGel^® solution to 37°C before mixing to lower its viscosity.

Mix the NexaGel^® solution gently with the cell medium and pipette slowly to avoid introducing air bubbles.

Briefly spin the mixing tube to remove any bubbles that may have formed.

No, changing the temperature does not trigger hydrogel formation in NexaGel^®. NexaGel^® remains in liquid form at room temperature. Hydrogel formation is initiated by mixing NexaGel^® with an ionic solution, such as cell culture medium. However, increasing the temperature can reduce the viscosity of the NexaGel^® solution. 

For the High Concentration hydrogels, it is not recommended. The concentration of the ionic molecule in medium would be much higher than the dilution solution, so diluting NexaGel^® with cell culture medium may cause the gel to form too quickly and chunky. 

In 3D cell culture, cells are encapsulated within the hydrogel matrix, allowing them to grow and interact in a three-dimensional environment. In contrast, 2D hydrogel coating involves applying a layer of hydrogel onto the bottom of the culture plate, with cells seeded on top of this layer. When using a thick 2D hydrogel coating, both the stiffness and the availability of functional ligands in the substrate are altered, which can influence cell behavior such as morphology and mobility. On the other hand, a thin 2D hydrogel coating applies only a very thin layer to the plate surface, modifying functional properties without significantly changing the stiffness of the underlying plastic.

Yes. Cells can be retrieved from the NexaGel^® system by applying the NexaGel^® Cell Recovery Solution. After recovery, the cells can be re-cultured for further growth using fresh NexaGel^®.

Typically, we recommend changing the cover medium every other day, similar to regular 2D cell culture. However, it depends on the experiment’s needs. Some experiments might require changing every 24 hours and some might not require changing for an entire week. 

Changing 100% of the cover medium might disrupt the hydrogel. We recommend adding additional fresh medium without removing the top medium for the first medium change. Afterward, change 50-80% of the cover medium.

Using the same number of cells is generally acceptable. However, the optimal seeding density can vary depending on the cell type and specific culture conditions. In many cases, using a slightly higher cell number and seeding the cells as small clusters rather than single cells can help improve cell survival.

The optimal final cell concentration may vary depending on the cell type. We recommend preparing the cell suspension at the following concentrations when working with NexaGel^®:

3D Cell Cultures:

For Ready-to-Use NexaGel^® hydrogels:

• Prepare the cell suspension at 1–2 × 10⁶ cells/mL.
• Mix the NexaGel® solution with the cell suspension at a 2:1 ratio (v/v).
• This results in a final cell concentration in the hydrogel of approximately 0.5–1 × 10⁶ cells/mL.

For High-Concentration NexaGel® hydrogels:

• Prepare the cell suspension at 0.5–2 × 10⁶ cells/mL.
• Mix the NexaGel® solution with the cell suspension at a 4:1 ratio (v/v).
• This results in a final cell concentration in the hydrogel of approximately 1–4 × 10⁵ cells/mL.

2D Hydrogel Coating:

We recommend a cell suspension concentration of 1–5 × 10⁵ cells/mL.

Yes, you can place the NexaGel^® mixture in an incubator rather than leaving it at room temperature. However, be aware that gel forms more slowly than at room temperature. For lower-concentration gels, this means a longer incubation time may be required for full stabilization. In contrast, higher-concentration gels or NexaGel^® Hydrogel Matrix are generally less affected by this and will still form gels effectively in the incubator.

Gel dissociation directly in 96-well plate is possible:

Assuming 50 uL of gel in a well of a 96-well plate. 

• Remove the cover medium, add 100 uL of pre-warm recovery solution (recommend MS04 organoid recovery solution for faster gel dissociation), mixing gel and solution by pipetting up and down for 5-10 times, add another 200 uL pre-warm cell recovery solution, mixing by pipetting up and down for 5-10 times.
• Keep the plate at 37°C for 5-10 mins (for strong gel, 15 min). If they have a plate shaker, use it during the 37°C incubation.
• Take the plate out and centrifuge the plate at room temperature.

The mixing ratio for the Ready-To-Use NexaGel^® is 2:1. 

For our High-concentration NexaGel^® it is 4:1. 

The mixing ratio is also tunable depending on the cell culture medium. For example, if the cell medium has a low ionic molecule concentration, such as RPMI, we recommend a 1:1 mixing ratio for ready-to-use hydrogel.

In most cases, allowing the gel to stabilize at room temperature for 10–15 minutes is sufficient. However, incubation time can vary depending on the temperature — lower temperatures generally lead to faster gelation. To ensure proper gel formation before adding the cover medium, a waiting period of 10–30 minutes at room temperature is recommended as a safe range.

Yes, NexaGel^® is compatible with various staining and imaging protocols, allowing detailed analysis of cell morphology and function.

Yes, cells embedded in NexaGel^® can be successfully fixed using PFA. Immunolabeling works well within the NexaGel^® matrix, allowing for clear visualization of cellular markers while preserving the 3D structure.

No, co-staining with crystal violet and DAPI is not recommended. Crystal violet staining involves methanol, which can interfere with DAPI staining and negatively impact cell imaging. Instead, we recommend using DAPI to stain the nuclei together with phalloidin to visualize the cytoskeleton and cytoplasm for clearer and more compatible results within the NexaGel^® system.

To image NexaGel^® within a cell culture insert, you can simply remove the insert from the cell culture insert plate and place it directly under the microscope for imaging. Alternatively, you can carefully cut out the membrane along with the hydrogel from the insert and proceed with imaging or further analysis.

Yes, nuclear staining agents such as DAPI are a great method to visualize cell invasion.

Yes, cells cultured in NexaGel^® can be used for molecular analyses following standard protocols. NexaGel^® is transparent and compatible with many molecular assays, allowing you to perform analyses directly within the hydrogel. Additionally, the hydrogel can be easily broken down using a homogenizer or ultrasonic processor, enabling efficient lysis of both the cells and the matrix for DNA or RNA extraction.

Fixation: Fix the hydrogel samples with 3.7% formaldehyde (v/v) for 1 hour at 37°C.

Washing: Wash the samples three times with D-PBS to remove residual fixative.

Dehydration: Dehydrate the samples using a graded ethanol series (0%, 30%, 50%, 70%, 85%, 95%, and 100%). Each ethanol concentration should be applied three times, for a total of 15 minutes per step.

Drying: Perform critical point drying to preserve the hydrogel structure.

Mounting and Coating: Mount the dried samples and sputter-coat them with a thin layer of gold.

Imaging: Scan the samples using scanning electron microscopy (SEM) to visualize the hydrogel structure.

This protocol helps preserve the delicate 3D structure of NexaGel^® for high-resolution SEM imaging.

Adjust the speed and force of liquid handling—use gentle pipetting to avoid disturbing the hydrogel.

During the first few washes, remove only 50–80% of the PBS or washing solution, and reserve complete removal (100%) for the final wash step.

Keep the plate and washing solutions cold, as lower temperatures help maintain the stiffness and structural integrity of the NexaGel^® hydrogel during handling.

We recommend fixating the hydrogels at 4°C, but high-concentration versions can also be fixed at room temperature.

Yes, NexaGel^® can be used for dome culture. We recommend using non-tissue culture treated plates to avoid potential detachment from the plates.

Non-treated culture plates are recommended to improve NexaGel^® adhesion to the plate.

Choose the NexaGel^® ORGANOID version based on the specific organoid type and desired mechanical properties.

Hydrogel Suitability for Organoid Types

Each version offers different mechanical strengths and bio-functional ligands to support various organoid cultures.

The mechanical strength of the different NexaGel^® ORGANOID hydrogels are in this order: 

NexaGel^® ORGANOID-3 > NexaGel^® ORGANOID-4 > NexaGel^® ORGANOID-2 > NexaGel^® ORGANOID-1.

NexaGel^® ORGANOID-3 is a great starting option, as many researchers have achieved successful results with this hydrogel for organoid culture.

Choosing the optimal NexaGel^® formulation depends on your specific research goals. Some researchers prioritize the formulation that yields the highest number of organoids for expansion, while others select the one that produces the largest and healthiest organoids for transplantation. Additionally, assessing key biomarkers can help evaluate important characteristics and confirm the quality of the organoids for your intended application.

Add a 3X concentration of your medium supplements (e.g., R-Spondin, Noggin, etc.) to the cell suspension before mixing with NexaGel^® at a 2:1 ratio. This ensures that the final hydrogel contains at least a 1X supplement concentration. (If desired, you can prepare a 5X supplement in the cell suspension to enhance initial cell survival and growth.)

To improve the initial survival rate of organoids, seed cells as small clusters rather than single cells and include a ROCK inhibitor (such as Thiazovivin).

If you wish to combine NexaGel^® with BME, add 10–20% BME to the cell suspension before mixing with NexaGel^®.

For culturing crypts, dilute NexaGel® with DI water at a 1:1 ratio before mixing with the cell suspension, as crypts tend to require lower gel strength.

Yes, organoids that grow in the NexaGel^® system can be passed.

The frequency of harvesting depends on the cell culture cycle, but typically organoids can be collected every 5 to 10 days.

No, it is not necessary to gradually decrease the percentage of Matrigel when transitioning organoids from a Matrigel-based culture to a xeno-free NexaGel^® culture. If you prefer to include Matrigel during the transition, you can add a small amount (less than 50%) to the cell suspension before mixing it with the NexaGel^® solution.

NexaGel^® Cell Recovery Solution is designed to recover intact 3D cells and organoids from both NexaGel^® and animal-based ECMs like Matrigel. It effectively dissolves the surrounding matrix, releasing cells from 3D encapsulation or 2D coating surfaces. However, it does not replace trypsin, as it cannot break cell–cell junctions or detach cells from plastic surfaces.

To obtain single cells, you can either:

Mix NexaGel^® Cell Recovery Solution with trypsin or another dissociation reagent at a 1:1 ratio, or

Use a two-step method—first, recover the intact 3D cells with NexaGel^® Cell Recovery Solution, then apply a dissociation reagent (e.g., trypsin) to break them into single cells.

NexaGel^® Cell Recovery Solution is specifically formulated to break down ionic-based crosslinks in hydrogels. It may also be effective in dissociating certain soft hydrogel networks. However, its effectiveness with GelMA, collagen, PEG-based hydrogels, or alginate will depend on the specific crosslinking chemistry of those materials. For hydrogels with covalent or strong physical crosslinks, additional or alternative enzymatic or chemical dissociation methods may be required.

Yes. Geltrex can vary in purity between batches, which may affect how easily it degrades. To improve recovery using NexaGel^® Cell Recovery Solution, follow these steps:

After removing the culture medium, wash the sample 1–2 times with PBS or NexaGel^® Cell Recovery Solution.

Add the NexaGel^® Cell Recovery Solution and pipette the mixture up and down several times — this step is key to speeding up gel breakdown.

Place the sample in an ice bucket and incubate for 2–5 minutes.

If working with 100% Geltrex, you may need to extend the incubation time up to 20 minutes.

To further eliminate gel residues, centrifuge the sample, discard the supernatant, and resuspend the pellet in fresh NexaGel® Cell Recovery Solution.

NexaGel^® Cell Recovery Solution is specifically formulated to recover intact organoids, not to dissociate them into single cells. If single-cell dissociation is required, you can either:

Add a cell dissociation reagent after harvesting the organoids from the NexaGel^® system.

or

Mix the dissociation reagent with NexaGel^® Cell Recovery Solution at a 1:1 ratio during the harvesting process.

Both incubation time and mechanical force influence the outcome of organoid recovery. Longer incubation times help dissolve the hydrogel more thoroughly, while mechanical actions — such as pipetting the mixture up and down — can accelerate matrix breakdown and fragment the organoids. For intact organoids, minimize mechanical stress; for smaller fragments or partial dissociation, controlled pipetting can be applied.

NexaGel^® is suitable for both 3D and 2D cultures; choose based on the experimental goals and cell type

Layer NexaGel^® with different cell types to create co-cultures or sandwich cultures, supporting complex interactions.

Spheroid formation typically occurs within 24-48 hours, depending on cell type and culture conditions.

Yes, NexaGel^® can encapsulate spheroids, allowing differentiation media to be applied for further development.

Yes, NexaGel^® hydrogels are highly suitable for creating 3D cell models used in drug or compound screening. NexaGel^® is compatible with high-throughput screening platforms, including automated liquid handling systems. Compounds can be either mixed directly into the hydrogel before gelation or added on top of the gel after cell encapsulation, allowing for efficient diffusion through the matrix.

Yes, NexaGel^® can be added directly on top of the cells or tissue slide.

Yes, NexaGel^® can be used to perform 3D transfection studies.

Yes, cell mobility on NexaGel^® can be observed in both 3D and 2D hydrogel coating cultures. NexaGel^® can also be used for spheroid invasion, wound healing, or 3D cell migration assays.

Yes, NexaGel^® is well-suited for spheroid invasion assay applications. Below is a general protocol you can follow:

• Seed cells into a well plate to form spheroids.
• Centrifuge the plate to encourage cell aggregation at the bottom.
• Remove most of the medium carefully (or initially add only a small volume to minimize removal later).
• Prepare the NexaGel^® hydrogel by mixing it with cell culture medium (without cells) at a 2:1 ratio (NexaGel^® : medium, v/v).
• Add the prepared hydrogel directly on top of the formed spheroids in each well.
• Centrifuge again briefly to ensure good contact between the spheroids and the hydrogel.
• Let the gel stabilize at room temperature for 10–15 minutes.
• Finally, add the cover medium on top for long-term culture and analysis.

This setup allows you to monitor spheroid invasion into the surrounding NexaGel^® matrix over time.

Injection volume varies based on application; consult specific protocols for guidance.

Cell number depends on the experimental design; ensure sufficient density for effective results.

Adjust the mixing ratio based on desired mechanical properties and cell viability; consult specific protocols.

No, maintain at room temperature unless specified by the protocol to prevent premature gelation.

NexaGel^® is our injectable hydrogel that can be injected into any part of the animal.

Yes, NexaGel^® allows for different injection sites, such as subcutaneous, orthotopic, IP, etc. Some difficult injections with an animal-based matrix can be performed smoothly with NexaGel^®.

We normally use 18-30 G needle size.

Following injection, the sol-gel transition occurs instantly, restoring the mechanical integrity of the hydrogel and effectively encapsulating the cells within the matrix. As a result, the hydrogel system provides excellent cell retention post-injection.

Unlike Matrigel, which rapidly solidifies at temperatures above 10°C, the NexaGel^®-cell mixture forms a soft, injectable hydrogel. Thanks to its unique shear-thinning and rapid self-recovery properties, the hydrogel remains injectable over extended periods—typically for several weeks or even months—provided that fresh culture medium is supplied and the embedded cells remain viable.

Yes, the successful cell type list is constantly growing.

NexaGel^® supports highly efficient tumor formation, achieving nearly 100% tumor formation rates with standard cell types and over 70% with more challenging ones. In most cases, tumor growth kinetics in NexaGel^® are comparable to or faster than those observed with Matrigel.

Tumor size and weight can be influenced by many factors. NexaGel^® is a fully synthetic, xeno-free hydrogel designed to support cells without altering their natural growth behavior. In contrast, Matrigel, being animal-derived, contains numerous undefined components and growth factors that can directly impact cell growth. Therefore, it's not unusual to observe slower tumor growth with NexaGel^® compared to Matrigel.

To potentially enhance tumor growth with NexaGel^®, you might consider the following strategies:

Increase the number of injected cells to stimulate stronger initial growth and promote faster tumor development.

Strengthen the gel structure by placing it on ice for 10–15 minutes before injection. This helps the cells remain clustered after injection, which can accelerate growth.

Boost supplements or growth factors in the cell suspension (e.g., FBS or other growth factors) up to 5X concentration to enhance cell proliferation post-injection.

For aggressive tumor types, an ulceration rate of approximately 10–20% is expected when tumor volumes exceed 500 mm³—this can be difficult to completely avoid.

However, certain technical factors may contribute to the development of ulcerations:

Intradermal implantation: Xenografts should be implanted subcutaneously (s.c.), allowing them to remain flexible and mobile beneath the skin. If the injection is fully or partially intradermal, the tumor may adhere to and invade the dermis, increasing the risk of ulceration.

Leakage at the injection site: If the cancer cell mixture leaks from the puncture after injection, ulceration may occur at the site. To minimize this:

Use a small gauge needle (27G or 30G).

Hold the syringe in place for a few seconds before withdrawing to allow the NexaGel^® mixture to start solidifying.

Replace the needle every 2–3 mice (or more frequently).

Clean any leakage with a sterile cotton pad, and gently pinch the skin for a few seconds to help seal the injection site.

Gel preparation: To reduce the risk of leakage, keep the NexaGel^®-cell mixture on ice before injection, or allow it to partially solidify to increase viscosity.

No, adding supplements to the hydrogel matrix is not required. For most xenograft applications, preparing cells in a PBS solution works exceptionally well with NexaGel^®.

NexaGel^® is also animal-origin-free, which allows researchers to manipulate the supplement in a gel-cell mixture if needed.

The choice of growth factors depends on your specific application and cell culture system. Common additives include BSA, HSA, or serum replacements.

If the use of serum is not an issue for your experiment, adding FBS can provide an additional boost to promote more robust cell growth.

No, there is no issue with bubble formation due to the low viscosity of NexaGel^®. There is an option to warm the medium and hydrogel solution before mixing them, as this can help reduce the viscosity.

No, the hydrogel matrix supports the cells, preventing them from sinking and ensuring uniform distribution.

Yes, NexaGel^® is an excellent delivery system for cell therapy. By injecting NexaGel^®, scientists can achieve better cell retention and higher cell viability.

Yes, NexaGel^® supports suspension cultures, providing a stable environment for cell growth.

NexaGel^® can form hydrogel-cell beads, supporting various experimental designs.

Yes, recovered cells/organoids can be reseeded into new culture systems immediately.

No, NexaGel^® is enzyme-free and neutral pH, ensuring compatibility with downstream applications.

Ensure correct temperature handling and increase pipetting/rocking cycles; add more recovery solution if needed.

Avoid prolonged exposure to recovery solution and ensure gentle centrifugation; minimize mechanical stress.

No, sufficient incubation time is necessary for effective ECM or hydrogel dissociation.

It is recommended to increase growth factors in the initial phase to ensure organoids receive sufficient nutrients. Regular medium can be used once cells are established, with initial supplementation of growth factors.

For sample requests, please contact our Sales Team via the following website: 

3D Cell Culture Matrices