Fig. 2: Scheme of the LED photo-crosslinking and 3D-printing section of the microfluidic/3D-printing device. The droplet train is transferred from the chip microchannel into a microtubing in a straight section with nearly identical inner channel and inner microtubing diameter. Further downstream, the microtubing passes an LED-section for fast photo cross-linking to generate the microgels. This section is contained in an aluminum encasing to avoid premature crosslinking of polymer precursor in upstream channel sections by stray light. Subsequently, the microtubing is integrated into a 3D-printhead, where the microgels are jammed into a filament that is directly 3D-printed into the scaffold.

세포가 함유된 마이크로겔의 온칩 제작 및 인-플로우 3D 프린팅
구성:하나의 통합 프로세스에서 칩에서 스캐폴드 재료까지

Vollmer, Gültekin Tamgüney, Aldo Boccacini
Submitted date: 10/05/2021 • Posted date: 11/05/2021
Licence: CC BY-NC-ND 4.0

바이오프린팅은 세포가 실린 스캐폴드의 제조를 위한 유력한 기술로 발전했습니다. 바이오잉크는 바이오프린팅의 가장 중요한 구성요소입니다. 최근 마이크로겔은 세포 보호 및 세포 미세 환경 제어를 가능하게 하는 매우 유망한 바이오 잉크로 도입되었습니다. 그러나 이들의 미세유체 제작은 본질적으로 한계가 있는 것으로 보입니다.

여기에서 우리는 안정적인 스캐폴드에 직접 유입되는 바이오프린팅과 함께 세포가 실린 마이크로겔의 미세유체 생산을 위한 미세유체 및 3D 인쇄의 직접 결합을 소개합니다. 방법론은 세포를 단분산 미세 방울로 연속 온칩 캡슐화하여 후속 유입 교차 연결을 통해 세포가 함유된 마이크로겔을 생성할 수 있으며, 이는 미세관을 종료한 후 자동으로 얇은 연속 마이크로겔 필라멘트로 끼이게 됩니다.

3D 프린트 헤드로의 통합으로 독립형 3차원 스캐폴드에 필라멘트를 직접 유입 인쇄할 수 있습니다. 이 방법은 다양한 교차 연결 방법 및 세포주에 대해 설명됩니다. 이러한 발전으로 미세유체학은 더 이상 바이오 제조의 병목을 초래하는 현상이 아닙니다.

Bioprinting has evolved into a thriving technology for the fabrication of cell-laden scaffolds. Bioinks are the most critical component for bioprinting. Recently, microgels have been introduced as a very promising bioink enabling cell protection and the control of the cellular microenvironment. However, their microfluidic fabrication inherently seemed to be a limitation. Here we introduce a direct coupling of microfluidics and 3D-printing for the microfluidic production of cell-laden microgels with direct in-flow bioprinting into stable scaffolds. The methodology enables the continuous on-chip encapsulation of cells into monodisperse microdroplets with subsequent in-flow cross-linking to produce cell-laden microgels, which after exiting a microtubing are automatically jammed into thin continuous microgel filaments. The integration into a 3D printhead allows direct in-flow printing of the filaments into free-standing three-dimensional scaffolds. The method is demonstrated for different cross-linking methods and cell lines. With this advancement, microfluidics is no longer a bottleneck for biofabrication.

Fig. 1: Three-dimensional schematic view of the multilayer double 3D-focusing microfluidic channel system, (b) control of droplet diameter via the Capiilary number Ca, and accessible hydrodynamic regimes for droplet production: squeezing (c), dripping (d) and jetting (e). The scale bars are 200 µm.
Fig. 1: Three-dimensional schematic view of the multilayer double 3D-focusing microfluidic channel system, (b) control of droplet diameter via the Capiilary number Ca, and accessible hydrodynamic regimes for droplet production: squeezing (c), dripping (d) and jetting (e). The scale bars are 200 µm.
Fig. 2: Scheme of the LED photo-crosslinking and 3D-printing section of the microfluidic/3D-printing device. The droplet train is transferred from the chip microchannel into a microtubing in a straight section with nearly identical inner channel and inner microtubing diameter. Further downstream, the microtubing passes an LED-section for fast photo cross-linking to generate the microgels. This section is contained in an aluminum encasing to avoid premature crosslinking of polymer precursor in upstream channel sections by stray light. Subsequently, the microtubing is integrated into a 3D-printhead, where the microgels are jammed into a filament that is directly 3D-printed into the scaffold.
Fig. 2: Scheme of the LED photo-crosslinking and 3D-printing section of the microfluidic/3D-printing device. The droplet train is transferred from the chip microchannel into a microtubing in a straight section with nearly identical inner channel and inner microtubing diameter. Further downstream, the microtubing passes an LED-section for fast photo cross-linking to generate the microgels. This section is contained in an aluminum encasing to avoid premature crosslinking of polymer precursor in upstream channel sections by stray light. Subsequently, the microtubing is integrated into a 3D-printhead, where the microgels are jammed into a filament that is directly 3D-printed into the scaffold.
Fig. 3: a) Photograph of a standard meander-shaped layer fabricated by microgel filament deposition printing. The lines have a thickness of 300 µm. b) photograph of a cross-bar pattern obtained by on-top deposition of several microgel filaments. The average linewidth is 1 mm. c) photograph of a donut-shaped microgel construct. The microgels have been fluorescently labelled by FITC-dextran to demonstrate the intrinsic microporosity corresponding to the black non-fluorescent regions, d) light microscopy image of a construct edge showing that fused adhesive microgels form a continuous, three-dimensional selfsupporting scaffold with intrinsic micropores.
Fig. 3: a) Photograph of a standard meander-shaped layer fabricated by microgel filament deposition printing. The lines have a thickness of 300 µm. b) photograph of a cross-bar pattern obtained by on-top deposition of several microgel filaments. The average linewidth is 1 mm. c) photograph of a donut-shaped microgel construct. The microgels have been fluorescently labelled by FITC-dextran to demonstrate the intrinsic microporosity corresponding to the black non-fluorescent regions, d) light microscopy image of a construct edge showing that fused adhesive microgels form a continuous, three-dimensional selfsupporting scaffold with intrinsic micropores.
Fig. 4: a) Scheme of the perfusion chamber consisting of an upstream and downstream chamber, perfusion ports, and removable scaffolds to stabilize the microgel construct during 3D-printing, b) photograph of a microgel construct in the perfusion chamber directly after printing and removal of the scaffolds, c) confocal microscopy image of the permeation front of a fluorescent dye, where the high dye concentration in the micropores can be clearly seen, d) confocal microscopy image of YFP-labelled HEK-cells within a microgel construct.
Fig. 4: a) Scheme of the perfusion chamber consisting of an upstream and downstream chamber, perfusion ports, and removable scaffolds to stabilize the microgel construct during 3D-printing, b) photograph of a microgel construct in the perfusion chamber directly after printing and removal of the scaffolds, c) confocal microscopy image of the permeation front of a fluorescent dye, where the high dye concentration in the micropores can be clearly seen, d) confocal microscopy image of YFP-labelled HEK-cells within a microgel construct.
Fig. 5: a) Layer-by-layer printing of microgel construct with integrated perfusion channel. After printing of the first layer, a hollow perfusion channel is inserted. Subsequently, the second and third layers are printed. b) The construct is directly printed into a perfusion chamber. The perfusion chamber provides whole construct permeation via flows cin and cout, as well as independent flow through the perfusion channel via flows vin and vout. c) Photograph of a perfusion chamber containing the construct directly after printing. The flow of the fluorescein solution through the integrated PVA hollow channel is clearly visible.
Fig. 5: a) Layer-by-layer printing of microgel construct with integrated perfusion channel. After printing of the first layer, a hollow perfusion channel is inserted. Subsequently, the second and third layers are printed. b) The construct is directly printed into a perfusion chamber. The perfusion chamber provides whole construct permeation via flows cin and cout, as well as independent flow through the perfusion channel via flows vin and vout. c) Photograph of a perfusion chamber containing the construct directly after printing. The flow of the fluorescein solution through the integrated PVA hollow channel is clearly visible.
Fig. 6: a) Photograph of an alginate capsule fiber formed after exiting the microtube. b) Confocal fluorescence microscopy image of part of a 3D-printed alginate capsule construct. The fluorescence arises from encapsulated fluorescently labelled polystyrene microbeads to demonstrate the integrity and stability of the alginate capsules.
Fig. 6: a) Photograph of an alginate capsule fiber formed after exiting the microtube. b) Confocal fluorescence microscopy image of part of a 3D-printed alginate capsule construct. The fluorescence arises from encapsulated fluorescently labelled polystyrene microbeads to demonstrate the integrity and stability of the alginate capsules.

Keywords

biomaterials, microgels, microfluidics, 3D printing, bioprinting

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