저자는 바 덴-뷔르템베르크 재단 "Bioinspired 소재 합성" 프레임 워크 (BioMatS-14)에 그들의 재정 지원에 대 한 감사 하 고 싶습니다.

1. 하이드로 겔 준비
<ol>
	<li>BSA의 200 mg 이온된 H2O를 20% (w/v) BSA 주식 (재고 솔루션 A)을 만드는의 1 mL와 함께 믹스.</li>
	<li>THPC 솔루션 (134 mg/mL)의 이온된 수를 만드는 THPC의 4.835 mL와 혼합 165 µ L 재고 솔루션 (재고 솔루션 B).</li>
	<li>KCSSGKSRGDS (1,111.1 g/mol) 펩 티 드 (또는 동등한 세포 접착 펩 티 드)의 1 밀리 그램의 무게 그리고 살 균 H2O 10 mg/mL 해결책 (재고 솔루션 C)를 100 µ L에 희석. 참고:이 단계는 선택 사항이 고만 경우는 히드로 셀 문화 응용 프로그램에 대 한 의미는 포함 되지 해야 합니다.</li>
	<li>96 잘 접시의 바닥을 제거 하 고 이동식 플라스틱 랩으로 대체. 참고: 여기에 사용 되는 번호판에 대 한 하단의 쉽게 제거할 수 있습니다 각 잘;의 하단에 압력을 적용 하 여 얇은 플라스틱 하단 단순히 밖으로 떨어질 것 이다.</li>
	<li>BSA 재고 솔루션의 믹스 100 µ L (A) 및 THPC의 100 µ L 재고 솔루션 (B) (선택 사항: 재고 솔루션 C 공업화, 세포 접착 hydrogels 4 µ L) 하이드로 겔의 200 µ L를 96 잘 접시에. 중 합 후 균일 한 하이드로 겔을 보장 하기 위해 5 번 이상 아래로 pipetting으로 구성 요소를 믹스.</li>
	<li>모든 hydrogels는 제대로 생산 될 때까지 약 10 분 동안 실 온 (RT)에서 96 잘 접시를 놓습니다.</li>
	<li>신중 하 고 천천히 접시의 아래쪽에서 플라스틱 포장을 제거 합니다.</li>
	<li>작은 스탬프를 사용 하 여 96 잘 접시에서 hydrogels 누르고 살 균 PBS, pH 7.4와 1.5 mL 튜브에 그들을 전송. 참고:는 hydrogels 약 4 mm의 직경 및 높이 8 m m의 원통형 모양이 있다.</li>
	<li>hydrogels 인산 염 버퍼 식 염 수 (PBS) 몇 개월까지 4 ° C에서 저장 합니다.</li>
</ol>
2. 동결 된 Hydrogels
<ol>
	<li>살 균 이온된 H2O의 500 µ L와 1.5 mL 튜브와 뚜껑을 제거. 주걱을 사용 하 여 1.5 mL 반응 관에는 hydrogels 전송.</li>
	<li>1.5 mL 튜브 단단히 파라핀 영화-에 적어도 3 층 각 유리병에 대 한 포장. 바늘을 사용 하 여 튜브에서 가스 릴리스 수 있도록 영화에 있는 작은 구멍을 뚫.</li>
	<li>다음 단계 중 하나를 수행 하십시오.
	<ol>
		<li>액체 질소는 물과 히드로 완전히 고정 되도록 5 분 동안 솔루션에 유리병을 전송 합니다. 즉시 제거 후 액체 질소에서 녹고에서 물자를 방지 하기 위해 건조 기 동결에 튜브를 전송 합니다. 참고:이 절차 결과 모 공에 대 한 10-15 µ m 반경에.</li>
		<li>천천히는 hydrogels 동결 하룻밤-20 ° C에서 튜브를 유지. 즉시 제거 후-20 ° C에서, 녹고에서 물자를 방지 하기 위해 건조 기 동결에 튜브를 전송 합니다. 참고:이 절차에 대 한 모에 결과 반지름 µ m 50-60.</li>
	</ol>
	</li>
	<li>24 h (와 후에 하이드로 겔 주위 물의 완전 한 증발), 동결 건조 기에서 그것을 제거 하 여 소재를 녹여. 참고: 기 공 크기는 confocal 레이저 스캐닝 현미경 검사 법 (단계 5, "하이드로 겔 시각화")와 함께 분석할 수 있습니다.</li>
</ol>
3. 입자 침
<ol>
	<li>1.1-1.5 단계에 설명 된 대로 하이드로 겔을 준비 합니다.</li>
	<li>직접 구성 요소를 혼합 후 추가 NaCl 포화 (36 mg/mL)이 발생할 때까지. 솔트 크리스탈 흰색 침전으로 솔루션에서 볼 수 있습니다 때까지 소금을 추가 합니다.</li>
	<li>통에 96 잘 접시를 전송 하 고 중 합 일어난다 (약 10 분) 때까지 흔들어. 1.7-1.8 단계에 설명 된 대로 hydrogels는 접시에서 제거 합니다.</li>
	<li>하이드로 겔 템플릿에서 모든 소금 elute 하 통 (20 rpm)에 살 균 물에 RT에서 적어도 24 h hydrogels 품 어. 몇 개월에 대 한 PBS에 4 ° C에서 hydrogels를 저장 합니다.</li>
</ol>
4. 채널 형성
<ol>
	<li>1 단계에서 설명한 대로 hydrogels를 준비 합니다. 히드로 협공을 사용 하 여 솔루션에서 제거 하 고 높게 흡수 성 종이, 과잉의 물을 제거 드라이 아이스의 블록 위에 두십시오.</li>
	<li>고정 30 하이드로 겔의 블록에서 조심 스럽게 분리. 하이드로 겔;를 손상 하지 않습니다 조심 스럽게 주걱을 사용 하 여 블록 다쳤어요. 1.5 mL 튜브에는 히드로 전송 하 고 37 ° c.에 하룻밤 건조</li>
</ol>
5. 하이드로 겔 시각화
<ol>
	<li>PBS의 10 ml에서 rhodamine B의 1 마그네슘을 diluting 하 여 rhodamine B 재고 솔루션을 준비 합니다. 0.001 mg/mL의 농도 도달할 때까지 PBS에서 rhodamine 재고 솔루션을 diluting 하 여 직렬 희석을 준비 (희석 비율: 100)</li>
	<li>스토리지 솔루션 (예를 들어, 단계 2.4에서에서.)에서 하이드로 겔을 제거 하 고 0.01 mg/mL rhodamine B 해결책의 1 mL 1.5 mL 튜브에 그것을 전송. 실시간에서 rhodamine B 솔루션에 하룻밤 히드로 얼룩</li>
	<li>다음 날, PBS (pH 7.4)와 적어도 3 h에 대 한 세척의 10 mL를 시각화 하는 히드로 전송.</li>
	<li>잘 µ-슬라이드 8에 히드로 전송 및 PBS와 그것을 커버.</li>
	<li>블레이드를 사용 하 여 (약 0.5 m m에 높이) 히드로에서 작은 조각을 잘라.</li>
	<li>Confocal 레이저 스캐닝 현미경를 사용 하 여 시각화 514의 파장에서 히드로 nm (목표: EC 계획-Neofluar 40 x / 1.30 오일 DIC M27, 비행기 스캔 모드: 514 nm, 및 확대/축소: 1 X).</li>
</ol>
6. 셀 문화 타당성
<ol>
	<li>멸 균 필터 모두 0.45 μ m 필터 솔루션 (A, B 및 C) 재고.</li>
	<li>1.1-1.4, 히드로 합 전에 셀 접착제 펩 티 드를 포함 하 여 단계에 설명 된 대로 하이드로 겔을 준비 합니다.</li>
	<li>구성 요소를 혼합 한 후 즉시 플라스틱 혼합물 µ-슬라이드 8에 하단 완전히 덮여 때까지 잘. 참고:이 단계 수행 되어야 합니다 신속 하 고 직접 부품, 후 중 합 슬라이드에 몇 분 안에 일어난다.</li>
	<li>문화 접착 세포와 통로를 표준 세포 배양 기술을 사용 하 여 단일 셀 서 스 펜 션. 미리 데워 진, 무 균 DMEM 세포 배양 소 태아 혈 청 (FBS, 10% (w/v)), 페니실린-스 (1% (w/v)), 그리고 불필요 한 아미노산 솔루션 (MEM, 1% (w/v)) 보충으로 셀을 전송 합니다.</li>
	<li>Neubauer 챔버 및 신중 하 게 피 펫 200 µ L 셀 (2 x 105 셀/cm2) 하이드로 겔 표면에 원하는 수의 계산 된 셀 계산.</li>
	<li>Μ-슬라이드 8 뚜껑으로 잘 커버 하 고 인큐베이터 (37 ° C, 5% CO2)에 그것을 전송. 적어도 4 h 37 ° c.에 품 어</li>
	<li>세포 부착의 적어도 4 h, 후 셀 메 마른 세포 배양 PBS의 200 µ L 두 번 씻는 다.</li>
	<li>RT에서 10 분에 대 한 3.7% (v/v) 포름알데히드의 200 µ L로 셀을 수정 하 고 PBS로 두번 세척 (~ 200 µ L). 참고: 포름알데히드를 처리할 때 적절 한 개인 보호 장비를 착용 하십시오.</li>
	<li>PBS로 두 번 5 분 세척에 대 한 0.1% 트리톤 X의 200 µ L 셀 permeabilize</li>
	<li>Phalloidin-rhodamine 셀 추가 하 여 PBS의 195 µ L methanolic 주식의 5 µ L 혼합 그것 셀에 실시간 얼룩에 20 분에 대 한 셀 어둠 속에 얼룩. PBS로 2 회 세척.</li>
	<li>514에 confocal 현미경을 사용 하 여 셀 접착 속성 조사 nm (목표: EC 계획-Neofluar 40 x / 1.30 오일 DIC M27, 비행기 스캔 모드: 514 nm, 및 확대/축소: 1 X). 적절 한 소프트웨어를 사용 하 여 이미지 분석 ( 테이블의 자료를 참조).</li>
</ol>
7. 하이드로 겔 속성
<ol>
	<li>붓기 비율입니다.
	<ol>
		<li>적어도 1 일 동안 37 ° C에서 hydrogels를 완전히 건조.</li>
		<li>각 히드로 무게 하 고 정확한 무게 합니다.</li>
		<li>1.5 mL의 PBS로 2 mL 반응 관을 채우십시오.</li>
		<li>이 2 mL 반응 관에는 히드로 전송 및 완전히 PBS에 몰두.</li>
		<li>PBS와 평형에 도달 하는 RT에 PBS에 적어도 이틀 동안은 히드로 둡니다.</li>
		<li>3 일 후, 솔루션에서는 하이드로 겔을 제거 하 고 히드로 표면에서 과잉의 물을 제거 하는 종이 티슈로 건조 합니다.</li>
		<li>무게는 하이드로 겔.</li>
		<li>다음 수식을 사용 하 여 붓기 비율 계산: 
		<img alt="Equation" src="/files/ftp_upload/55813/55813eq1.jpg"> 여기서 Wd 는 말린된 젤 (7.1.2 단계.)의 무게와 Ws 젖은 젤 (단계 7.1.7)의 무게입니다. 붓기 비율 %를 100으로 곱하면 됩니다.</li>
	</ol>
	</li>
	<li>pH 및 온도 안정성입니다.
	<ol>
		<li>15 mL 반응 관에 PBS의 5 mL을 전송.</li>
		<li>NaOH와 HCl. 가져와 적절 한 온도 (예, RT, 37 ° C, 또는 80 ° C) 솔루션 솔루션 적절 한 산도 (예를 들어, : pH 2, 7, 또는 10)을 조정 합니다.</li>
		<li>적절 한 솔루션으로 안정성에 대 한 조사 하는 히드로 전송. 필요한 경우 pH를 재조정 하 고 참고: 모든 hydrogels 해야 합니다 수 완전히 불이 시점에서 (붓기 비율 참조) 재료의 붓기 때문에 무게의 잘못 된 해석을 방지 하기 위해.</li>
		<li>특정 시간 간격 후는 하이드로 겔 (예를 들어, 최대 2 일 동안 각 시간) 솔루션에서 제거, 과잉의 물을 제거 하 고 그것의 무게를 매우 흡수 성 종이 휴지로 건조.</li>
	</ol>
	</li>
	<li>효소 저하입니다.
	<ol>
		<li>300 U 트립 신 펩 신, 제조업체의 지침에 의하여의 재고 효소 솔루션을 준비 합니다.</li>
		<li>적절 한 솔루션을 (예를 들어, 단계 1.8에서에서) 히드로 전송. 참고: 모든 hydrogels 해야 합니다 수 완전히 불이 시점에서 무게의 잘못 된 해석을 방지 하기 위해.</li>
		<li>특정 시간 간격 후는 하이드로 겔 (예를 들어, 각 시간) 솔루션에서 제거, 과잉의 물을 제거 하 고 그것의 무게를 매우 흡수 성 종이 휴지로 건조.</li>
	</ol>
	</li>
</ol>

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저자 들은 아무 경쟁 금융 관심사 선언 합니다.

The production of macroporous matrices can be beneficial to many different fields. It has high technical and economic potential due to the defined structure of the hydrogel and the ability to control and tune specific material properties. However, the introduction of supramolecular structural elements, such as pores or channels, to a 3D template might influence the overall properties of a material, such as the swelling ratio or the stiffness. This can result in the undesired decomposition, degradation, or breakdown of the network, thus rendering the material useless or simply inapplicable for the desired application12. Proteins possess a known potential for application in hydrogels due to their defined structure, their intrinsic biocompatibility (because they are derived from natural sources), and their many available modification sites (e.g., reactive amino-, hydroxyl- or carboxyl- groups). However, many protein-based hydrogels lack sufficient mechanical stability compared to their synthetic counterparts16. Because the introduction of large 3D architecture may lead to the complete structural disintegration of a hydrogel, the feasibility of methods to tune 3D structures must be critically evaluated to obtain the desired material properties and to achieve the full potential of the protein material. This was one reason for the evaluation of the freeze-drying and particle leaching processes and of the effect those procedures had on the resulting properties of the protein hydrogels in this study.
A critical point while working with hydrogels is the visualization of the materials on the macro-scale. Quite often, electron microscopy-based methods are used to examine the 3D structure of a hydrogel28. While doing this, it is important to consider that many procedures used to prepare the sample for electron microscopy might interact with the material and significantly alter its properties and integrity (e.g., the freezing of the sample). To overcome this problem, confocal laser scanning microscopy was used here to evaluate the produced 3D architectures and the size and distribution of the pores within the hydrogels. As opposed to electron microscopy, only a simple staining step in the native hydrated environment of the swollen state of the material is necessary to analyze the micro-scale structure of the matrix.
Freeze-drying is a very well-established method in soft matter research and biochemistry. It allows for the production of pores of a certain size and regular distribution throughout the matrix due to ice crystal formation in the network. By applying very low pressure (i.e., ~0.05 mbar) and temperatures below 0 &#176;C, the ice sublimates from the material, leaving pores behind without further disintegrating the surrounding network. In the swollen state, pores of about 10 to 70 &#181;m were detected in the template (Figure 1), with a narrow size distribution for smaller pores and a wider distribution for larger pores. Those sizes are within a suitable range for most cell culture-related applications, like hematopoietic stem cell incorporation3 and the ingrowth of osteoids and fibroblasts21. However, this method also has certain limitations. For some applications, the creation of larger pores might be beneficial22. The size limit that could be achieved with freeze drying was approximately 70 &#181;m in this material, but larger pores have already been developed by different groups21.
The dried hydrogels must be handled with care; due to their fragile structure, they tend to break apart when slight pressure is applied to the dried material. The critical step in the preparation of the material is the polymerization. Both components must be mixed properly and quickly to guarantee the production of a uniform hydrogel. If the hydrogels are not mixed immediately, a partial polymerization might occur, leading to an uneven pore distribution within the matrix.
Another technique that is well-established for the modification of synthetic polymer materials is the so-called particle leaching. Solid but dispersible particles are incorporated into the material prior to the gel-sol transition and are later eliminated from the polymerized material by changing external conditions, leading to the solubilization of the particles13,29. Paraffin and gelatin can be melted by increasing the temperature29, while crystalline particles, like sugars and salts, are dissolved by changing the buffer composition or dilution13. For this protein-based material, the radii of the pores could be changed 10-fold within the 3D matrix. Untreated salt crystals created the largest pores, with radii about 50 to 70 &#181;m (Figure 2). One limitation of this method is the maximum size of the pores: above 70 &#181;m, a considerable population of the pores exhibited fractures in their backbones, thus destabilizing the matrix. To overcome this problem, salt crystals were ground with a mortar and pestle, and significantly smaller pores, without damaged structural backbones, were produced. To improve the method, the crystals can be sieved to achieve a specific size3. Another critical step is the saturation of the solution: the hydrogels must be completely liquid when adding the salt. If the hydrogel is already partly polymerized, uneven pores will appear in the mesh, or the gel will break. If too little salt is added, the solution might be too diluted, causing no pores appear. To overcome this problem, a test series must be made prior to the experiment to find the exact amount of salt that is necessary for saturation.
Channels within a material can be useful for certain applications that require scaffold material (e.g., the ingrowth of axons into a template)11, but they are often difficult to produce in soft materials due to the lack of stability and rigidity. We established a process that includes the use of a block of solid carbon dioxide to produce linear-oriented channels within the protein hydrogel, with a length of several hundred microns and a radius of approximately 20 &#181;m (Figure 3). During their formation, the time that the hydrogel remains on the block of dry ice is essential and must be evaluated individually for each material. Short freezing times do not lead to the formation of structures, while longer freezing times break the network. When removing the material, damage to the hydrogel can be avoided by pressing a spatula against the block of ice and scratching some of the ice off the block along with the hydrogel.
All procedures influence the structure of the material and may thus severely reduce the beneficial properties of the material. To address this effect, different basic gel parameters were evaluated. The swelling ratio is one of the most important features with respect to the use of a hydrogel in cell culture, as it directly correlates with the free water content, an essential factor for the growth and survival of cells within a 3D template. All treated hydrogels were dried and brought to swelling equilibrium prior to the measurements. The swelling ratios for the different methods varied from 534% to 1,153% (Table 1). The free water content is crucial to prevent the accumulation of harmful intermediates in the environment of the cell. At the same time, nutrients, growth factors, and oxygen should be able to diffuse rapidly throughout the gel to fulfill the demands of a growing and dividing cell7. With both freeze-drying and salt leaching, a high water content could be achieved for the protein-based macroporous hydrogels. Furthermore, the swelling ratio has a significant influence on the differentiation of stem cells within a matrix and can be further improved by incorporating PEG into the material8. This is easy to do in protein hydrogels with a variety of target sites.
Many hydrogels are intended for use in biotechnological fields, such as cell culture2, tissue engineering30, or transplantation medicine31. The material must deal with harsh external conditions, including temperature or pH changes and the presence of degradation agents, like proteases or catalytic active substances. Especially in materials intended for use in the human body, such as coatings for medical devices32 or injectable hydrogels33, the systems might mutually interact with the host system, thus influencing their stability and possibly limiting their use. As a variety of methods to tune 3D architecture severely influence the material stability, all procedures used in this study were investigated for their influence on the mechanical and chemical properties of the hydrogel. The reaction of the BSA protein hydrogel mainly depends upon the applied stimuli. For example, a temperature increase from RT to 37 &#176;C or a pH shift into the acidic range did not alter the protein stability, while a shift to basic conditions reduced the residual weight after a week (Table 1). More importantly, none of the procedures used here influenced the stability of the matrix in comparison to one another (Table 1).
For many applications, a cleavable backbone is desirable. Most protein hydrogels are biodegradable. However, many hydrogels require extended decomposing times, as agents must often work from the outside to the inside of the material. This further leads to uneven degradation patterns, resulting in the complete breakdown of the network after a certain, undefined time. Untreated BSA hydrogel was degraded after several days, with the complete breakdown and disintegration of the network after a certain time. Hydrogels treated with freeze-drying or particle leaching approaches degraded in a predictable manner within hours when in the presence of enzymes, as shown in Figure 4. For macroporous hydrogels, enzymes can freely diffuse throughout the matrix and cleave the backbone of the whole template, leading to an degradation and predictable degradation times. The degradation time was found to be dependent upon the sizes of the pores within the material. For example, hydrogels frozen at -20 &#176;C, with an average pore radius of 60 &#181;m, could be degraded to half of their weight within 3.2 h using 300 U of pepsin. Hydrogels frozen at -196 &#176;C, with a significantly smaller average pore radius of 10 &#181;m, had a half-life of 6.5 h at the same pepsin concentration (Table 1).
A major concern for cell culture applications is the adhesion potential of the matrix. Many materials facilitate adhesion due to their inherent adhesion properties, while other systems must be provided with proper cellular adhesion through the incorporation of cell-adhesive structures. One prominent example of guaranteed cell adhesion is a cell-adhesive peptide, with RGD one of the most prominent and well-characterized34. Protein-based materials are normally easy to modify due to the vast amount of accessible target sites within the template. To prove the general feasibility of this BSA-based protein hydrogel for cell culture applications, the material was functionalized with a cell-adhesive RGD peptide. To facilitate the process, a specific KCSSGKSRGDS peptide was designed with a primary amine in the lysine moiety, which can be targeted by the crosslinking agent during polymerization. A549 and MCF7 cells were grown on the surface of the protein hydrogel and showed distinctive adhesion after 24 h of growth, with high viability16. The growth of the cells and the strong adhesion, which could not be reversed by washing with PBS, show the viability of the cells on the matrix and the general feasibility of the produced material for cell culture application. The direct incorporation of the peptide is both an advantage and a limitation of this system: on the one hand, it simplifies the procedure and eliminates the need for an additional reaction step. On the other hand, many different cell-adhesive peptides have been developed and used over the last decade5, and some of them bear primary amines in their structure. Those amines will readily react with the hydroxy groups of the linker molecule, thus potentially disabling the adhesion properties of the peptide. The peptide must be chosen according to this system. Another option is to add the peptide of choice after the polymerization. This way, it can be linked to different moieties in the protein backbone, whichever is best for the desired application.
For further use in 3D cell culture, the system must be optimized and adapted for each specific cell line. By changing the protein or crosslinker concentration, the properties can already be tuned, especially the mechanical properties of the material. The presented methods offer a range of tools for the manipulation of hydrogels, with no need for special equipment and with low costs. The techniques maintain the general properties of the protein-based hydrogel, making them useful for the initial evaluation of 3D cell culture applications.

Hydrogels는 많은 양의 물 바인딩 수 불용 성 3 차원 네트워크를 형성 하는 물자. 이러한 자료는 살아있는 세포에 대 한 우수한 환경 조건을 제공할 수 있습니다. 현재, 3 차원 하이드로 겔 구조의 세대에 그들의 화학 및 물리적 특성에 맞게 프로세스 개발에 관심이 증가 하고있다. 이 달성 되 면 세포의 성장 및 세포 행동의 조작에 대 한 템플릿이 생성된1,2,,34될 수 있습니다. 이러한 3 차원 구조 뿐만 아니라 기존의 2 차원 방법, 하지만 그들은 보다 더 자연스럽 고 현실적인 환경 또한 공개 새로운 가능성을 만들 줄기 세포의 성장에 대 한 또는 종양 모델5. 다른 재료는 젤6의 기 공 크기에 주로 의존 하는 특성의 범위를 소유한 다. 모 공 세포 문화 응용 프로그램, 조직 공학 및 줄기 세포의 감독된 성장에 중요 한 역할을 한다. 예를 들어, 매트릭스를 통해 확산 하는 산소와 영양분 그리고 충분 한 양의 세포7에 도달 할 수 있어야 합니다. 다른 한편으로, 유해한 대사 산물, 최대한 빨리 제거 되어야 한다 고 세포 성장 위한 충분 한 공간이 사용 가능한7수 있어야 합니다. 따라서, 재료의 기 공 크기, 속성 심각 하 게 영향을 미칠 행렬의 잠재적인 혜택 및 가능한 응용 프로그램. 재료의 특성에 따라, 다른 세포-성장 과정; 신경 구조의 형성을 포함 하 여 3 차원 세포 배양에서 발생할 수 있습니다. 성장과 분화의 피부 나 뼈 세포; 그리고 hepatocytes 또는 섬유 아 세포2,3,,89,10,11같은 특별 한 줄기 세포 라인의 감독된 성장. 또 다른 중요 한 포인트는 물질의 가능한 응용 프로그램에 영향을 미치는 외부 자극12쪽으로 그것의 안정성입니다. 예를 들어는 히드로 세포 배양 또는 인체에 기계적 무결성을 유지 해야 합니다.
최근 몇 년 동안, 강화, 3D 셀 문화 hydrogels에 연구와 많은 연구 시스템13의 3 차원 구조를 해결 하기 위해 실시 했다. 화학적 합성된 구성 요소 구성 Hydrogels 가장 일반적으로 쉽게 합성 및 화학적 수정 수 있고 그들은 높은 안정성을 전시 하기 때문에 조사 (주 외., 2011 리뷰 참조)5. 그러나, 단백질에는 많은 유익한 속성이:으로 소위 "정밀 고분자," 그들은 생체; 그들은 정의 길이; 그들은 상대적으로 쉽게 수정; 그리고 그들은 많은 수의 대상 사이트14,15. 이와 관련, 많은 분야에서 응용 프로그램에 대 한 매우 구체적인, 혁신적인 구조를 생성할 수 있습니다. 본이 연구에서는 단백질 기반 하이드로 겔16 자료의 3 차원 구조에 영향을 잘 설립 방법의 능력을 보여 주기 위해 사용 되었다. 또한, 능력과 기 공 세대에 적용도 조사 했다.
많은 다른 기술이 간단한 방법 및 재료 과학의 다른 분야에서 정교 하 고, 고도로 전문화 된 기술 모두를 포함 하 여 3 차원 구조를 수정할 수 있습니다. 광범위 한 기술을 잘 정의 된 구조17를 생성 하는 전기의 사용 이다. 충전된 섬유는 전기 분야에 의해 해결책에서 가져온 고 산소에 노출 시 응고. 이 방법에서는, 몇몇 미크론까지 여러 나노미터의 범위에서 섬유를 생산 수 있습니다. 추가 기술을 크기, 구조, 그리고 매트릭스 내의 숨 구멍의 배포를 조정 하는 소프트 리소 그래피, 사진 평판, 유체역학 초점, 전기 살포 바이오 인쇄18,,1920. 이러한 기술의 중요 한 결점은 특정, 비싼 장비 및 특수 화학 제품 또는 자료에 대 한 의존도. 또한, 이러한 기술을 경험 종종 단백질 기반 재료를 직접 양도 이며 화학 물질 및 방법의 많은 셀 호환 되지 않습니다.
다른 한편으로, 많은 기술을 쉽고 저렴 적용 하 고 재현 하 그들을 만드는 특별 한 장비에 의존 하지 마십시오. 구조 조작에 대 한 광범위 한 방법은 용 매 캐스팅21,,2223입니다. 입자 중 합 반응 전에 추가 되 고 솔루션 포화 homogenously 배포 됩니다. 중 합 후 희석 또는 pH 변화, 같은 조건의 변화를 모는 재료 내에서 유지 하는 동안 입자의 solvation에 지도 한다. 소금, 설탕, 파라핀, 젤라틴, 분필, 등이 기술에 사용 되는 화학 물질 저렴 하 고 쉽게 사용할 수 있다 동결에 부 어 hydrogels 동결 됩니다. 진공에서 액체 단계의 후속 승화는23,,2425수행. 네트워크에서 물 승화 만큼 부드러운 재료의 특정 3D 구조를 유지 하는. 가스 거품, 동안에 중 합, 젤21내 숨 구멍을 떠나는 해결책 가스와 스트리밍됩니다. 크기와 숨 구멍의 분포는 가스 흐름에 따라 조정할 수 있습니다.
형성 단백질 히드로, BSA는 tetrakis (hydroxymethyl) phosphonium 염화 물 (THPC) 1 차 아민 4 무장 링커 분자26의 hydroxy 그룹 사이 공유 결합의 형성에 대 한 있도록 Mannich 형 반응에서으로 반작용 했다. 반응 발생 가능한 유해한 중간체 물질의 과도 한 씻기에 의해 제거 됩니다.
이 연구에서는 조작 하 고 숨 구멍의 크기에 맞게 다른 기술로 BSA 기반 소재를 치료의 가능성을 보여 줍니다. 각 기술을 사용할 수 있습니다 어떤 실험실에 전세계, 특별 한 장비는 필요 하다. 또한, 다른 매개 변수를 같은 붓기 비율, 효소 분해성, pH 안정성, 온도 감도, 검사 그리고 서로에 비해 특히 존중 다른 기술의 영향에 3D 아키텍처의 생성에. 마지막으로, 자료 했다 기능성 세포 배양 물질의 가능한 응용 프로그램을 조사 하기 위해 셀 접착제 펩 티 드와 함께. 두 개의 서로 다른 모델 셀 라인 사용 되었다: A549 및 MCF7.

<table border="0" cellpadding="0" cellspacing="0" width="1102">
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			<td height="20" style="height:20px;width:479px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:253px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">10010023</td>
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			<td height="20" style="height:20px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (high glucose)</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>11140-050</td>
			<td></td>
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			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>10270-106</td>
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			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>15140122</td>
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			<td height="20" style="height:20px;">MEM Nonessential Amino Acid Solution</td>
			<td>Sigma Aldrich</td>
			<td>M7145-100ML</td>
			<td></td>
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			<td height="20" style="height:20px;">Trypsin EDTA 0.05 % Phenol Red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300062</td>
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			<td height="20" style="height:20px;">Ethanol 99.8 %, verg&#228;llt</td>
			<td>&#214;lfabrik Schmidt</td>
			<td>2133</td>
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			<td height="20" style="height:20px;">NaCl&#160;</td>
			<td>Carl Roth&#160;</td>
			<td>9265.1</td>
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			<td height="20" style="height:20px;">Albumin Fraction V</td>
			<td>Carl Roth&#160;</td>
			<td>3854.2</td>
			<td></td>
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			<td height="20" style="height:20px;">THPC</td>
			<td>Sigma Aldrich</td>
			<td>404861-100ML</td>
			<td>Toxic</td>
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		<tr height="20">
			<td height="20" style="height:20px;">0.1 % Triton X 100</td>
			<td>Sigma Aldrich</td>
			<td>X100-100ML</td>
			<td>Slightly toxic</td>
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			<td height="20" style="height:20px;">Phalloidin-rhodamine&#160;</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>R415</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">3.7 % Formaldehyde&#160;</td>
			<td>Life Technologies / Thermo Fisher&#160;</td>
			<td>F8775-25ML</td>
			<td>Toxic</td>
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			<td height="20" style="height:20px;">Rhodamine B</td>
			<td>Sigma Aldrich</td>
			<td>81-88-9</td>
			<td></td>
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			<td height="20" style="height:20px;">Filtropur S 0.2,&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>2 83.1826.001&#160;&#160;&#160;</td>
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			<td height="20" style="height:20px;">&#181; slide 8 well</td>
			<td>Ibidi GmbH</td>
			<td>80826</td>
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			<td height="20" style="height:20px;">KCSSGKSRGDS peptide</td>
			<td>UPEP Ulm</td>
			<td>Custom sysnthesis</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Ethanol 99.8 %, verg&#228;llt</td>
			<td>Carl Roth&#160;</td>
			<td>K928.5</td>
			<td></td>
		</tr>
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			<td height="20" style="height:20px;">Falcon 5 ml Polysterene Round-Bottom Tube&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>62.547.254&#160; &#160;&#160;</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Tubes 50 ml&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>62.547.254&#160; &#160;&#160;</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Tubes 1,5 ml&#160;&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>72,690,001</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Tubes 2 ml&#160;&#160;</td>
			<td>Sarsted Ag und Co.</td>
			<td>72,691</td>
			<td></td>
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			<td height="20" style="height:20px;">CELL CULTURE MICROPLATE, 96 WELL, PS, F-BOTTOM</td>
			<td>Greiner</td>
			<td>655073</td>
			<td></td>
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			<td height="20" style="height:20px;">FreezeDryer Epsilon 1-6D,&#160;</td>
			<td>Christ, Osterode am Harz, Germany</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Confocal Laser Scanning Microscope&#160;</td>
			<td>Carl Zeiss AG, Oberkochen, Germany</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Zen Software Version 2012 Sp1, black edition, 407 version 8,1,0,484</td>
			<td>Carl Zeiss AG, Oberkochen, Germany</td>
			<td></td>
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			<td height="20" style="height:20px;">GSA Imaga Analyzer Software, GSA Image Analyzer, GSA, Version 419 3.8.7</td>
			<td>GSA GmbH</td>
			<td></td>
			<td></td>
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easy manipulation of architectures in protein-based hydrogels for cell culture applications

Hydrogels 셀 문화 응용 프로그램 때문에 높은 수 화 셀 환경을 제공 하는 그들의 능력에 대 한 유망 자료로 인정 받고 있습니다. 3 차원 서식 파일의 필드는 자연적인 기질을 그 물질의 잠재적인 정체 때문 상승 이다. 단백질 기반 hydrogels가 있습니다 특히 유망한 functionalized 쉽게 수 있기 때문에 조정 가능한 물리 속성으로 정의 된 구조를 얻을 수 있습니다. 그러나, 천연 재료를 사용 하 여 셀 문화 응용 프로그램에 대 한 macroporous 3D 템플릿의 생산 종종 합성 물질의 그들과 비교 된 그들의 약한 기계적 성질에 의해 제한 됩니다. 여기, 다른 방법을 macroporous 소 혈 청 알 부 민 (BSA)를 생산 하기 위해 평가 했다-하이드로 겔 시스템, 반경에서 10 ~ 70 µ m의 범위에서 조정 가능한 기 공 크기를 기반으로. 또한, 몇 백 미크론 긴은이 단백질 기반 자료 채널을 생성 하는 방법 설치 되었다. 모 공, 기 공 크기의 영향 비율, pH, 온도 안정성, 그리고 효소 저하 행동, 붓기 등의 소재 속성에 생산 하는 여러 가지 방법은 분석 했다. 기 공 크기 hydrogels confocal 레이저 스캐닝 현미경 검사 법을 사용 하 여 네이티브, 부푼 상태에서 조사 되었다. 셀 문화 응용 프로그램에 대 한 타당성 단백질 시스템 및 두 개의 모델 셀 라인의 세포 접착제 RGD 펩타이드 수정을 사용 하 여 평가 했다: 인간 유방암 세포 (A549)와 adenocarcinomic 인간의 치경 기저 상피 세포 (MCF7).

Hydrogel development has become one of the most prominent fields in material research-related biological studies, with thousands of entries indexed in scientific research archives. Although the behavior of many systems is well studied, the manipulation of 3D networks, especially of sensitive protein-based materials, is often a major issue in material science. Another commonly underestimated challenge is the correct measurement of the native structure of a material using cryo electron microscopy. This is because the sample preparation (i.e., drying) process often changes the hydrogel properties. To overcome this problem, the material samples here were analyzed using confocal microscopy, a method that allows for the characterization of the material in its native, water-swollen state. Rhodamine B can bind to the hydrogel backbone via electrostatic interaction and facilitates the visualization of the material. To investigate the feasibility of a freeze-drying approach to modify this protein-based system, hydrogels were subsequently polymerized and frozen at 196 &#176;C in liquid nitrogen and at -20 &#176;C. The results show a clear influence of the freezing temperature on the resulting pore sizes. At -196 &#176;C, small pores with a radius of about 10 &#181;m and a narrow size distribution were produced. On the other hand, freezing at higher temperatures leads to the production of materials with much larger pores, whose radii are between 50 and 70 &#181;m, as seen in Figure 1. The slow freezing process leads to the formation of larger ice crystals, which results in bigger pores within the material after the sublimation of the ice.
Another technique for pore generation, which is well-described for synthetic materials, is particle leaching. Solid particles like paraffin, gelatin, salt, or another solid-state material, are incorporated into the hydrogel prior to solidification. They are then eluted from the solid hydrogel by changing external conditions, such as temperature, Ph, or buffer composition by dilution. The BSA-based hydrogel was polymerized in the presence of high salt concentrations. After the sol-to-gel transition, salt crystals were removed from the material by adding an excess of water to dilute the crystals. This was followed by the confocal analysis of the resulting materials. The size of the salt crystals was changed by grinding NaCl with a mortar and pestle. As shown in Figure 2, the pore size can be tremendously changed using this method. Depending upon the crystals used (untreated or ground for a certain time), pore sizes from 10 to 70 &#181;m could be produced. Appropriately sized pores are essential for many applications that feature the incorporation of cells into a hydrogel. However, more sophisticated structures might be needed when the creation of more complex structures is desired (e.g., to provide structural guidance for neuronal cells along a gradient). Complex architectures can often only be produced with special equipment (e.g., spin coating, electrospinning, lithographic, or bioprinting techniques). However, the production of linear channels can be realized with a simple freeze-drying approach and the use of dry ice. The channels produced have radii of about 20-30 &#181;m and lengths of several hundreds of micrometers, as shown in Figure 3.
Another crucial feature of hydrogels is the biodegradability of the template matrix, both in vivo and in vitro1. Protein hydrogels offer the advantage of being biodegradable. However, many systems lack fast degradation due to the restricted target sites within the material. Proteolytic agents must work their way from the outside of a material to the inside, resulting in very slow degradation times. In contrast, macroporous hydrogels are normally well-diffusible. This allows enzymes to target protein structures throughout the whole template simultaneously, which greatly reduces degradation times. For the presented protein-based hydrogel, the proteolytic degradation time depends mainly on the pore sizes within the matrix. A representative degradation kinetic is shown in Figure 4. Pore generation reduces the time from several days to a few hours for trypsin and pepsin degradation, as shown in Table 1.
The main benefit of hydrogels is the high water content within the materials, which is desirable to mimic the extracellular matrix27. The swelling ratio represents the amount of water a gel can absorb and hold after drying and is a valid indicator of the free water content of a macroporous hydrogel. Furthermore, diffusion depends strongly upon the free water content and is required for the transport of nutrients towards the cells and the removal of toxic metabolites. The swelling ratio correlates directly with the pore size within the material, as shown in Table 1. By changing the pore size using the demonstrated methods, it is possible to alter the diffusion behavior in the template and thus to influence cell fate by manipulating their feed.
The stability of a hydrogel to external stimuli, such as pH and temperature changes, severely limits or expands the possible applications of a system. Particularly for application in a cell culture-related area, resistance to changes in the external parameters is essential, as cells and cell culture matrix mutually influence their properties and behavior (e.g., the acidification of the medium might lead to hydrogel degradation, and hydrogel degradation leads to cell release or death)22. To demonstrate the feasibility of the methods described here for pore generation, all hydrogel stabilities were determined for increasing temperatures (37 &#176;C and 80 &#176;C) and changing pH values (pH 2, 7, and 10). The residual weights of the hydrogels, which were treated using different methods, are summarized in Table 1. In conclusion, the presented protein-based hydrogels are stable over a wide range of conditions, while the residual weights of the hydrogels at high temperatures only reduce gradually. For the swelling ratio, it is important to consider that a partial unfolding might occur at higher temperatures, even if most BSA molecules should be held in place by the four-armed linker.
Whenever a macroporous hydrogel is intended for use in cell culture, the adhesion properties of the material play a crucial role in the individual application. Some materials bear inherent adhesion properties (e.g., heparin, short, extracellular matrix-derived peptide structures, or fibronectin)5. For those that do not have these features, the possibility to efficiently modify the material is desirable in order to introduce cell adhesion properties. One major advantage of protein hydrogels is the presence of a variety of accessible functional groups on the surface, which can be targeted by specific reactive linker molecules. Another option is the incorporation of cell adhesive peptides during polymerization, which can further facilitate the handling of the material and the production process, as only a single step is necessary to produce cell-adhesive materials. By using a four-armed, amine-reactive crosslinking agent26, the cell adhesion mediating peptide (in this case, RGD) can be directly co-polymerized during hydrogel formation. This procedure results in the proper adhesion of the cells, as shown in Figure 5. Two model cell lines, human breast cancer cells (A549, CCL-185) and adenocarcinomic human alveolar basal epithelial cells (MCF 7, HTB-22) were used to investigate the general feasibility of using this modified hydrogel in 3D cell culture. Both cell lines showed very good adhesion potentials on the modified hydrogels.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55813/55813fig1.jpg" /> 
Figure 1: Freeze-drying the hydrogels. After hydrogel polymerization, the gels were transferred to -196 &#176;C (liquid nitrogen) or -20 &#176;C. The material was stained with fluorescent rhodamine B and analyzed with confocal laser scanning microscopy. The size and distribution of the pores within the material were analyzed with imaging software and were plotted for the different temperatures. On the right side, representative pictures of the materials frozen at (A) -196 &#176;C and (B) -20 &#176;C are shown. The error bars represent the standard deviation. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55813/55813fig2.jpg" /> 
Figure 2: Hydrogel particle leaching. Salt crystals are ground with a mortar and pestle for (A) 10 min, (B) 2 min, (C) 1 min, and (D) 0 min. Both hydrogel components are mixed, and the solution is saturated with salt crystals. After the polymerization of the materials, salt is eluted from the material by diluting the template in large amounts of deionized water. The material is stained with fluorescent rhodamine B and analyzed with confocal laser scanning microscopy. The size and distribution of the pores within the material were analyzed with imaging software and plotted for the different grinding times. Images A to D show the pores within representative hydrogels, where salt crystals of varying sizes were used. The error bars represent the standard deviation. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55813/55813fig3.jpg" /> 
Figure 3: Channel formation. The polymerized hydrogels were transferred to a block of dry ice for 30 s. This was followed by the sublimation of the ice crystals from the structure at 0.05 mbar and -85 &#176;C. The material was stained with fluorescent rhodamine B and analyzed with confocal laser scanning microscopy. The channels were several hundred microns long and had diameters of about 20 &#181;m. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55813/55813fig4.jpg" /> 
Figure 4: Enzymatic degradation. Representative degradation pattern of a macroporous hydrogel treated using a salt-leaching approach with ground salt crystals (10 min). Degradation took place in solution with 300 U trypsin and pepsin and was compared to an untreated hydrogel in PBS by measuring the residual weight of the material every hour. The error bars represent the standard deviation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55813/55813fig5.jpg" /> 
Figure 5: Cellular behavior on hydrogels. To investigate the potential of the material in cell culture, hydrogels were functionalized with a cell-adhesive RGD peptide. The hydrogels were polymerized into a &#181;-slide 8 well and seeded with 2 x 105/cm2 A549 and MCF7 cells. The cells were allowed to grow and adhere to the material for 24 h. After fixation with 3.7% formaldehyde and permeabilization with 0.1% Triton X-100, the cells were stained with cell-specific phalloidin-rhodamine and were visualized with inverted confocal laser scanning microscopy at a wavelength of 514 nm. Scale bars = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55813/55813fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2"></td>
			<td rowspan="2">Salt leaching</td>
			<td rowspan="2">Salt leaching (grinded salt, 10 min)</td>
			<td>Frozen at&#160;</td>
			<td>Frozen at&#160;</td>
			<td rowspan="2">Gradient freezing</td>
			<td rowspan="2">Non-porous hydrogels</td>
		</tr>
		<tr>
			<td>-20&#176;C</td>
			<td>-196&#176;C</td>
		</tr>
		<tr>
			<td>pH 2 [%]</td>
			<td>95.3 &#177; 3.8</td>
			<td>96.2 &#177; 3.3</td>
			<td>94.4 &#177; 3.4</td>
			<td>96.2 &#177; 5.2</td>
			<td>89.7 &#177; 4.3</td>
			<td>69.5 &#177; 4.4</td>
		</tr>
		<tr>
			<td>pH 7 [%]</td>
			<td>95.6 &#177; 2.3</td>
			<td>94.4 &#177; 4.2</td>
			<td>95.3 &#177; 5.6</td>
			<td>94.2 &#177; 3.2</td>
			<td>94.1 &#177; 3.2</td>
			<td>98.2 &#177; 1.6</td>
		</tr>
		<tr>
			<td>pH 10 [%]</td>
			<td>82.1 &#177; 4.4</td>
			<td>86.1 &#177; 3.2</td>
			<td>76.3 &#177; 5.5</td>
			<td>83.2 &#177; 4.3</td>
			<td>84.2 &#177; 4.5</td>
			<td>42.3 &#177; 4.1</td>
		</tr>
		<tr>
			<td>RT [%]</td>
			<td>97.4 &#177; 4.4</td>
			<td>95.4 &#177; 0.42</td>
			<td>91.3 &#177; 2.2</td>
			<td>94.3 &#177; 4.1</td>
			<td>97.1 &#177; 1.9</td>
			<td>99.1 &#177; 2.2</td>
		</tr>
		<tr>
			<td>37&#176;C [%]</td>
			<td>95.3 &#177; 4.2</td>
			<td>97.4 &#177; 0.4</td>
			<td>93.2 &#177; 3.3</td>
			<td>96.2 &#177; 1.9</td>
			<td>95.3 &#177; 4.3</td>
			<td>98.3 &#177; 3.4</td>
		</tr>
		<tr>
			<td>80&#176;C [%]</td>
			<td>81.2 &#177; 4.4</td>
			<td>83.6 &#177; 4.5</td>
			<td>84.2 &#177; 4.9</td>
			<td>83.5 &#177; 3.4</td>
			<td>91.4 &#177; 8.1</td>
			<td>70.2 &#177; 6.2</td>
		</tr>
		<tr>
			<td>Swelling ratio [%]</td>
			<td>1153 &#177; 110</td>
			<td>534 &#177; 45</td>
			<td>1312&#160; &#177; 91</td>
			<td>834 &#177; 78&#160;</td>
			<td>823 &#177; 163</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin [h]</td>
			<td>4.5 &#177; 0.23</td>
			<td>7.4 &#177; 0.29</td>
			<td>3.2 &#177; 0.21</td>
			<td>6.5 &#177; 0.13</td>
			<td>4.2 &#177; 0.13</td>
			<td>55.0 &#177; 2.48</td>
		</tr>
		<tr>
			<td>Pepsin [h]</td>
			<td>3.1 &#177; 0.19</td>
			<td>4.0 &#177; 0.22</td>
			<td>2.4 &#177; 0.13</td>
			<td>3.8 &#177; 0.14</td>
			<td>3.5 &#177; 0.19</td>
			<td>46.5 &#177; 3.02</td>
		</tr>
	</tbody>
</table>
Table 1: Hydrogel properties. To investigate the influence of the pore formation methods used here (top line), different types of hydrogels were investigated for their pH and temperature stabilities, swelling ratio, and enzymatic degradation pattern (left column). For pH values of 2, 7, and 10 and temperatures of RT, 37 &#176;C, and 80 &#176;C, the average residual weights of the hydrogels after 7 days are displayed as percentages. For enzymatic degradation, the half-lives (h) of the materials in 300 U trypsin and pepsin are shown. For the swelling ratios, the water uptakes based upon the dried gels are shown are percentages.

Watch this Scientific Journal Video about Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications at JoVE.com

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

단백질 기반 hydrogels 3 차원 구조를 조작 하는 다른 메서드 여기 소재 속성에 대해 계산 됩니다. 세포 접착 펩 티 드와 함께 macroporous 네트워크는 공업화 하 고 두 개의 서로 다른 모델 셀 라인을 사용 하 여 세포 배양에서 그들의 타당성을 평가.

셀 문화 응용 프로그램에 대 한 단백질 기반 Hydrogels 아키텍처의 쉬운 조작

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

easy-manipulation-architectures-protein-based-hydrogels-for-cell

Nanyang Technological University

Research

JoVE Journal

Biochemistry

6.7K 조회수.  Ulm University. 단백질 기반 hydrogels 3 차원 구조를 조작 하는 다른 메서드 여기 소재 속성에 대해 계산 됩니다. 세포 접착 펩 티 드와 함께 macroporous 네트워크는 공업화 하 고 두 개의 서로 다른 모델 셀 라인을 사용 하 여 세포 배양에서 그들의 타당성을 평가.

비디오: Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

양적 응용 프로그램에 대한 균일 MALDI 시료의 제조

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

연구

생화학

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

질량 분석 기반 식별 RNA 바인딩 영역에 대 한 샘플 준비

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

마그네틱 핀셋으로 G-quadruplexes의 단일 분자 조작

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

분해 활동에 대 한 화면에 Fluorogenic 펩 티 드 분열 분석 결과: 코로나 바이러스에 대 한 응용 프로그램 스파이크 단백질 활성화

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

차에서 파생된 새로 설립된 세포 현탁액 배양에서 6개의 전신 살충제의 물질대사에 관한 연구(동백나무시넨시스 L.) 잎

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

탠덤 질량 분석과 결합 된 액체 크로마토그래피를 사용하여 사카로 미세화세세비시아의 세포 지질의 정량분석

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

성숙한 쌀의 횡단 단면(오리자 사티 라 L.) 파이펫 팁을 사용하여 전자 현미경 이미징을 스캐닝하기 위한 커널

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

탠덤 질량 분광과 결합 된 액체 크로마토그래피를 사용하여 사카로미세스 Cerevisiae의 정량적 Metabolomics

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA 메틸트랜스퍼라제 억제제를 스크리닝하기 위한 연속 형광 기반 엔도뉴클레아제 결합 DNA 메틸화 분석

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.