Liquid-Like Solids (LLS®)

The First Microgel-Based 3D Cell Culture Medium

Description

Aurita's patented 3D cell culture medium constitutes a new class of materials. Unlike other hydrogel-based 3D mediums which polymerize into a solid, preventing further handling, LLS® is comprised of soft, granular microgels which allow it to retain its liquid-like handling indefinitely while also providing a stable, adaptive support structure for biological samples when undisturbed.

LLS® is fully defined, being comprised of a synthetic hydrogel, and can be tailored to meet the demands of most biological niches with tunable stiffness and surface bio-conjugations. LLS® is also designed to closely match the refractive index of culture media, which when coupled with its ability to suspend biological samples in position indefinitely, makes it highly desirable for high-resolution microscopy applications.

Liquid-Like Solids (LLS®) vs. Existing 3D Culture Techniques

The unique granular structure of LLS® allows it to incorporate many of the fluid handling and self-healing benefits of liquid culture mediums, while also providing the physical support of scaffold and ECM mediums.

Workflow Considerations

LLS® is designed to be compatible with an impressive variety of biological samples, high-fidelity microscopy applications, liquid handling devices, and a wide selection of existing assays.

Transparent

LLS® is comprised almost entirely of water, making it an excellent support medium for imaging samples in situ where fidelity and low optical distortions are critical.

Self-Healing

Upon yielding, the microgels which comprise LLS® shift to accommodate motion and newly deposited materials; when stress is removed, the microgels quickly settle to support delicate structures.

Liquid Handling

LLS® mediums have an extremely low yield stress in their solid state, and will shear and flow like classical fluids when pipetted or poured into a new vessel.

Tunable

The hydrogel composition of LLS® can be tailored to achieve a wide range of desired mechanical properties, including stiffness, particle size, or yield stress.

Permeable

The interstital space between LLS® enables free diffusion and fluid flow when paired with a Darcy Plate®, vastly improving transport over solid mediums like Matrigel® or hydrogels.

Synthetic Formulation

The chemical composition of LLS® is fully-defined and consistent between batches, with no unknown biological factors or animal-derived components.

Surface Bio-Conjugation

Surface conjugation of LLS® microgels provides additional environmental cues for biological samples and can be achieved with a variety of ECM proteins.

Simple to Use

LLS® is pre-polymerized, requiring only a brief equilibration with cell culture media before use. Centrifuging LLS® collects it at the bottom of the tube, where it can then be mixed cells.

Liquid-Like Solids (LLS)®

Highlighted Features and Applications

Designed for Microscopy

LLS nearly matches the refractive index of cell culture media, preventing optical distortions and facilitating clear visualization and high-fidelity imaging of delicate structures and cellular interactions in situ. Because LLS acts as a solid when undisturbed, samples remain in place for as long as the experiment requires.

Stromal Cell Ready

Stromal cells, including fibroblasts, immune cells, and endothelial cells, can be freely introduced into the local microenvironment to interact with the cells of interest, providing valuable insight into characteristics such as tumor growth, immune evasion, and response to therapies.

Tailored Microenvironment

LLS is comprised of soft, granular microgels that are bio-conjugated with ECM proteins to mimic the local tissue niche. This combination of mechanical stimuli, ECM proteins, and inherent porosity are readily tunable to meet the needs of a particular sample or experimental objective.

Real-Time Cell Tracking

Cells freely traffic through the interstitial spaces of the LLS microgels, enabling cell tracking, cell fate, and cell-cell interactions to be quantified in real time. Identify distinct cell behaviors in the vicinity of diseased tissues, characterize immune cell killing and exhaustion rates, or capture rare events in situ as they occur.

Always Accessible

No enzymatic or chemical digestion is required to liquify LLS in preparation for sample suspension or retrieval. The simple act of pipetting fluidizes the material, making sample collection remarkably straightforward.

Freeform BioPrinting

The unique rheological properties of LLS enable it to both freely accommodate the motion of a printing tip while also supporting the delicate structures once they are composed. Coupled with perfusion, which does not disturb the microgel bed, samples can effectively be cultured during the printing process.

Darcy Plates®

Aurita's Suite of Perfusion-Enabled 3D Cell Culture Devices

In the 1850’s French engineer Henry Darcy established an equation to describe fluid flow through a porous medium: Darcy’s Law. The Darcy Plate® platform abides by this law to provide passive perfusion of liquid cell culture media through the interstitial spaces of the granular LLS® 3D culture medium, emulating the interstital flow which permeates and supports live tissues in vivo.

Several unique Darcy Plates® are available to meet the demands of various applications, but they each achieve this passive flow in a largely similar manner. A microporous membrane separates feed media and sample reservoirs, and an induced pressure gradient drives cell culture media flow through the membrane and supported LLS® microgel bed. Flow rates typically decay over time, but are reset simply by refilling the feed reservoir and aspirating the collected effluent.

Darcy Plate® 3D Culture vs. Spheroid Culture Plates

Spheroid culture plates, typically comprised of an array of round-bottom wells, are excellent for generating uniform samples but critically lack the environmental cues necessary for translatable results.

Uniform contact with microgel particles over the entire surface of the sample.

Points of Contact

Single point of contact with the bottom surface of the dish.

Tunable, covering the physiological range for a variety of tissue niches.

Stiffness

Fixed, and many orders of magnitude greater than that of soft tissue.

Cells freely traffic through the interstitial spaces of LLS® and interact with samples.

3D Co-Cultures

Cells settle to the bottom without support, and will contact larger samples only at the bottom.

Thin coverslips and transparent membranes facilitate high-fidelity microscopy.

Imaging

Contoured surfaces introduce optical distortions for round-bottom plates.

Generate tens of thousands of spheroids, per well, in the interstitial spaces between LLS® microgels.

Spheroid Generation

Most are capable of generating one spheroid in each well.

Bioconjugating the surfaces of the LLS® microgels further improves their mimicry of ECM.

ECM Context

Limited to liquid culture medias, or cells will fail to settle and aggregate.

Passive perfusion continuously replaces depleted culture media and washes out harmful metabolites.

Nutrient and Waste Transport

Some natural convection may occur, but otherwise diffusion-limited.

LLS® facilitates printing in situ, permitting the arrangement of samples in each well at all times.

BioPrinting

Cells settle to the bottom without support, preventing any real spatial arrangements.

Darcy Perfusion Plates®

Highlighted Features and Applications

Passive Flow

Convection in cell culture enhances the transport of nutrients, dissolved gases, and signaling molecules through 3D mediums, mitigating nutrient depletion and local metabolite accumulation. Darcy perfusion devices can achieve this passively, requiring no external pumps or controllers.

Non-Destructive Washout

Effectively clear cytokines, DAMPs, metabolic waste, viral particles or other molecules by perfusion. The unidirectional flow means washed out molecules can be diluted and removed in the effluent without disturbing the sample positioning.

In Situ Dosing

Perfusion continuously exchanges the culture media in the microenvironment of your samples, facilitating the controlled introduction and dosing of a variety of solutes including therapy compounds, nanoparticles, viruses, and proteins.

High-Density Cell Culture

Culture and grow cells at extraordinary densities, 10 million+ per millileter, making these plates ideal for viral selections, biologic production, or spheroid generation. Perfusion flow enables long-term culture at high-density without needing to passage.

High-Fidelity Imaging, In Situ

The Darcy 24-well imaging insert incorporates an optically transparent membrane and thin coverglass to facilitate high-fidelity microscopy during perfusion culture. This enables a host of exciting, in situ, imaging-based assays.

Industry Standard Formats

Many Darcy perfusion devices adhere to industry-standard microplate formats, ensuring compatibility with various automated systems, liquid handling devices, and incubators commonly used in research settings.

Technology

Liquid-Like Solids (LLS)®

Darcy Perfusion Plates®