架橋PEGハイドロゲル中の細胞の写真 – カプセル化する方法について説明する。カプセル化されたマウスインスリノーマ(MIN6)集合内の低酸素シグナルが蛍光マーカーシステムを用いて追跡されます。このシステムは、ハイドロゲル骨格と細胞表現型の変化と低酸素シグナル伝達の相関関係にあるセルの連続検査が可能になります。
In Diabetes mellitus type 1, autoimmune destruction of the pancreatic β-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host’s immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact β-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80° C as many 150 μL aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL.
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1α for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.
ここで紹介するメソッドは、非生理的条件の最小限の使用とPEGハイドロゲル中の細胞のカプセル化のための迅速かつ簡単な技術を提供しています。 PEGは、その生体適合性と修正を容易にするために非常に有用な封止材料を表します。光活性溶液中でPEGの比率の単純な変化は、例えば、孔径によって、機械などの圧縮率などのプロパティ、およびトランスポートのプロパティを調整することができる。また、PEGを簡単に側鎖の付加によって変更されます。 PEGハイドロゲルは、従って、in vitroでの研究のための有望な臨床デバイスと柔軟なプラットフォームの両方を表す
PEG -カプセル化された細胞の低酸素状態を追跡するための方法も提示されている。このメソッドは、低酸素症検出の簡略化のためと目的の細胞を犠牲にする必要性を回避するのに便利です。テクニックは、その私たちを作る様々な条件で細胞のさまざまな種類のに適用してもよい幅広いefulness。例えば、幹細胞の分化のための手がかりとして、低酸素は、幹細胞の微量培養に追跡することができる。しかし、このメソッドは、分散した細胞は、後で集約されているセルのシステムまたはシステムを分散させるために適用することができます。また、蛍光シグナルの検出には大きいか密度の高い組織では難しいかもしれません。
The authors have nothing to disclose.
寛大にMIN6細胞を供給するためのコロラド大学、ボルダーのクリスティAnseth研究室に感謝します。このプロジェクトの資金は、NSFによって提供されています。
Name of the reagent | Company | Catalogue number | Comments (optional |
PEG | Sigma-Aldrich | 309028-500G | |
Methacrylic Anhydride | Sigma-Aldrich | 276685-100ML | |
Microwave | Emerson | MW8784SB | |
Vortexer | Scientific Industries | SI-A236 | |
Methylene Chloride | Sigma-Aldrich | D65100-1L | |
Diethyl Ether | Sigma-Aldrich | 346136-1L | |
Dialysis Tubing | Spectrum | 132640 | |
Laboratories | |||
Freezer | |||
Lyophilizer | Labconco | 7670521 | |
Vacuum pump | Welch | 8917Z-01 | |
Irgacure 2959 | Ciba-Geigy | 029891301PS04 | |
HBSS | Mediatech | 21-022-CV | |
Syringe Filter | VWR | 28145-477 | |
RPMI 1640 | Mediatech | * | *custom formulation |
FBS | PAA Laboratories | A15-351 | |
Penicillin-Streptomycin | Mediatech | 30-002-CI | |
Amphotericin B | Mediatech | 30-003-CF | |
Incubator | Thermo Scientific | 3597 | Napco Series 8000 WJ w/ O2 suppression |
Trypsin EDTA | Mediatech | 25-052-CI | |
Orbital Shaker | VWR | 12620-926 | |
UV Lamp | Sanyo Denri | FLR40SBLB/M | Holds two 40W, 365nm blacklight blue UV bulbs |
Centrifuge | Eppendorf | 5811 000.010* | *order number. Model 5810 R |
Microscope | Nikon | TI-ND6-PFS | With filterset for 556nm excitation/ 586nm emission |