PODS® Technology

 

 

 

Introduction

PODS® technology addresses the issue of protein fragility by placing proteins in a protective protein crystal lattice. This lattice stabilizes cargo proteins even at high temperatures. In the presence of proteases secreted by cells, pores develop in the lattice which allows the cargo protein to slowly escape. This gradual, sustained release maintains bioavailability at steady-state levels (zero-order kinetics). The crystals can also be readily used for patterning by attachment to surfaces or suspension at specific positions in hydrogels.   PODS® nanocrystals have a wide range of applications either in-vitro (2D or 3D culture) or in-vivo.

 

 

 

 

How are PODS® made?

PODS® contain a cargo protein that is encased within a microcrystal formed by self-assembly of polyhedrin protein. The process of generating these crystals all occurs inside the insect expression cell and results in perfect cubic structures about 0.2 - 5 microns across. The PODS polyhedrin crystal shell also protects the active protein during purification, ensuring the cargo protein is not denatured and is therefore correctly folded and functional. Even under the light microscope, you can see the cubic structure of the polyhedrin crystal. Empty PODS crystals are transparent but refract light when loaded with cargo. For most purposes, this does not interfere with imaging. 

 

Storage Stability

Following purification, PODS® proteins can be stored long term either dry or in liquid or hydrogel or another bio-scaffold, even at 37°C where 70% bioactivity is retained after 6 months. The important point is that the other material is protease-free.

 

Sustained release, steady state bioavailability

The PODS® crystals slowly release cargo through pores formed in the polyhedrin as it is degraded by proteases released by cells. Independent, peer-reviewed analysis has shown that a result of this sustained-release, which results in an optimal concentration zone, cells experience less stress and are consequently healthier.

 

PODS® can be readily positioned to act locally 

PODS® can be easily attached and immobilized to 2D surfaces and 3D scaffolds. The possibilities are limited only by our imagination. PODS can be used either naked or incorporated into bio-scaffolds and then positioned by hand or using an instrument, such as a 3D cell printer.

 

 

Attributes of PODS® growth factors:

Benefits and applications of PODS® growth factors:

 

Comparison of standard growth factors and PODS® growth factors

 

Standard vs. PODS® growth factors

Standard

PODS®

Readily soluble   Slowly soluble in the presence of proteases
Degrades even in sterile PBS Stable in PBS
Heat-labile Heat-stable over long periods
Difficult to localize Readily localized - readily form gradients
✓ Bioactive Bioactive
Difficult to incorporate into biomaterials Readily incorporates into biomaterials
Requires frequent replenishment Growth factor constantly replenished from store within PODS crystals 
$ to $$$$  - economical to very expensive $ - economical

 

 

 

  

Read PODS® support literature

See more Application Notes

 

Watch to learn more.

 

PODS® protein crystals explained by Dr Christian Pernstich

 

Watch how protein gradients can be generated with PODS®

 

 

Frequently Asked Questions

How big are PODS® crystals?

PODS® crystals are cubic and typically 0.2-5 microns in size with a modal size of 1-2 microns. 

What is a PODS® crystal's buoyant density?

PODS® co-crystals are slightly heavier than water and will settle on the surface of a culture vessel. Care should be taken when aliquoting since PODS® crystals will sink to the bottom of a tube within a few minutes. The majority of PODS® crystals will remain in suspension for up to 60 min in a 20% glucose solution (or a solution of similar density).

How stable are PODS® crystals?

We have tested a variety of conditions to assess their impact on the integrity of PODS® crystals. PODS® crystals are stable in high temperatures. They are highly stable when stored in solutions between pH 6-8. Above pH 10, PODS® crystals lose their stability and dissolve rapidly (in a few hours). PODS® crystals are stable in standard cell culturing temperatures for extended periods of time (>60 days).

How is the active protein released?

PODS® protein cargo is released by proteases. Proteases may be derived from components of the solution (e.g. serum) or secreted by nearby cells. Therefore the culture system affects the amount of growth factor available in solution. In contrast to gel-encapsulated proteins (using hydrogels such as PLGA) PODS® crystals do not produce an initial burst release of cargo proteins. For naked PODS® crystals in cell culture, peak release in once culture system typically occur at day 2, then gradually diminishes.  Peak-availability is not the same as peak-release-rate and depends on a number of factors including the rate of protease released by cells and the inherent stability (half-life) of the cargo protein.

Is it possible to modify the release profile?

The release of active proteins into cell culture occurs over a period of 1-3 weeks. This release period can be extended if the PODS® crystals are combined with a scaffold, which acts as a barrier to protease activity. Release rates will also be influenced by the presence of proteases in the scaffold. Bovine collagens, for example, will contain proteases.

Will PODS® crystals adhere to plastics?

PODS® crystals will adhere to untreated plastics and are therefore ideal for micro-patterning and scaffold functionalization. For sub-aliquoting, we recommend using low adherence plastics, such as low protein binding centrifuge tubes. 

Why are PODS® proteins sold by numbers of crystals?

PODS® active proteins are incorporated into the polyhedrin crystal within the insect cell. Consequently, it is not possible to determine the amount of active protein that is present in a sample by measuring optical density. When PODS® crystals are expressed, both polyhedrin proteins and active proteins are expressed from within a single vector and both utilize the same promoter sequence. Therefore, the ratio of active to polyhedrin protein will be constant. The size distribution of PODS® crystals is also constant. Consequently, the number of crystals has been adopted as the unit of quantification for PODS®. It has been estimated that 50x106 PODS® crystals typically generates a peak concentration in the medium equivalent to 3.3 µg of standard recombinant protein.

How many PODS® crystals should be used?  

PODS® crystals provide a depot of active proteins which are steadily secreted. As stated above, in one experiment it has been estimated that the biological activity of 50x106 PODS® crystals generates the same peak dose as 3.3 µg of standard recombinant protein in a LIF-dependent mouse ESC culture system. However, at 5 days following the start of seeding the PODS™ crystals, more than 50% of these peak levels are still present in the culture system. Ultimately, the amount of PODS™ crystals that are optimal for a particular experiment should be determined empirically, using 50x 106 PODS® crystals equivalence to 3.3 µg of standard growth factor as a good starting point.

How often should media and PODS® crystals be replaced in the culture system?

The frequency of media change depends on (1) the speed with which nutrients are exhausted or degraded and (2) the speed with which toxic metabolites accumulate. In most cell culture systems, the stability of growth factors is an over-riding issue which drives media replenishment. However, particularly in cells with high levels of metabolic activity, other factors will eventually become important once the stability issue has been addressed. We have maintained growth-factor-dependent cultures for several weeks without needing to add new PODS crystals.

Are PODS® crystals transparent?

PODS® without any cargo are isotropic crystals which do not refract light. However any cargo protein impacts on this property such that they will refract light. PODS® crystals generally do not interfere with the generation of images using imaging techniques such as phase-contrast and may also interfere with an assay that utilizes the measurement of optical density. 

Can PODS® crystals have a physical impact on cell behaviour?

Physical features on a culture surface may impact the behaviour of certain cells, particularly at high densities. If PODS™ crystal topology is a concern, PODS® crystals may simply be incorporated into a hydrogel surface coating. Phagocytic cells (such as macrophages) will ingest PODS®. We have not observed any effect on the health of these cells. However, if this is a concern, the PODS crystals may be incorporated into a gel. Custom made Matrigen™ Softwell plates (flat hydrogels of defined elasticity) containing PODS® crystals are available from Cell Guidance Systems. 

Do PODS® proteins have post-translational modifications? 

PODS® crystals are made in insect cells. PODS™ active proteins, therefore, contain most of the post-translational modifications that are also found in mammalian cells. 

Are PODS® crystals immunogenic?

The polyhedrin protein has been tested many times in-vivo and, although antibodies are generated against polyhedrin, an obvious inflammatory response has not been observed.  Lack of an inflammatory response for foreign proteins is not without precedent: silk fibroin protein from the silkworm B. mori is commonly used for surgical stitching and Botulinum toxin (Botox) is widely injected in cosmetic procedures.

Are PODS® proteins equivalent to standard growth factors?

The stability of standard recombinant growth factors varies significantly. The most labile, such as FGF-1, have a sera half-life measured in minutes limiting their utility. The stability of PODS® growth factors that are encased in PODS® crystals is much longer but once a molecule is released will have the same half-life as their standard recombinant counterparts. This may be advantageous for 3D applications as it reduces the path length

The amount of available growth factor in a culture system is a function of the speed of release from the crystal and the subsequent stability. In the first few hours of a culture containing only newly added PODS® crystals, there is little growth factor protein available for the cells. Significant amounts of growth factor protein are available after one day. The initial lag of protein availability may be corrected if necessary by adding a small amount of standard recombinant growth factor.

We are interested in developing therapeutic applications, can you help us?

We're always happy to explore the possibilities. We already have several therapeutic collaborations in place. Please contact us at info@cellgs.com if you plan to develop a therapeutic application.