本發明係關於一種量測細胞聚落之裝置及其方法，尤指一種可於細胞生長為細胞聚落(cell colony)的過程中，透過即時量測電阻抗值而獲得細胞生長資訊之裝置及其方法。The present invention relates to a device for measuring cell colony and a method thereof, and more particularly to a device and method for obtaining cell growth information by measuring an electrical impedance value in real time during cell growth as a cell colony .
近十年來，全球因癌症致死人數逐年攀升，癌症預防及癌症治療也成為各個國家備受重視之議題，並紛紛投入大量資源於癌症研究上。In the past decade, the number of cancer deaths worldwide has risen year by year. Cancer prevention and cancer treatment have also become the subject of great attention in various countries, and they have invested a lot of resources in cancer research.
因致癌因子誘發基因突變等因素，使正常細胞生長失去正常調控，而轉變為癌細胞。正常細胞根據其組織來源，具有特定之最大分裂次數，且細胞彼此相互接觸後，即停止生長分裂；然而，有別於正常細胞，癌細胞在適當的環境下，仍可持續進行細胞分裂，且癌細胞生長不會因細胞間相互接觸而停止，因此癌細胞過度生長分裂後將形成三維聚落(3D colony)。Due to factors such as carcinogenic factors-induced gene mutations, normal cell growth loses its normal regulation and turns into cancer cells. Normal cells have a specific maximum number of divisions according to their tissue source, and when the cells contact each other, the growth and division are stopped; however, unlike normal cells, the cancer cells can continue to undergo cell division under appropriate circumstances, and The growth of cancer cells does not stop due to mutual contact between cells, so the cancer cells will form a three-dimensional colony after overgrowth and division.
為能使癌症研究更近似於生理狀態，體外細胞培養亦從單層培養(monolayer culture)發展成三維培養(3D culture)，三維培養係將細胞與特定比例之培養膠體，即培養基與膠之混合物混合後進行培養，因膠體提供固態環境，使癌細胞得以形成三維聚落。然而，癌症相關研究，包括建立藥物篩選平台及探討致病機制等，均需準確量化所培養之細胞已獲得細胞生長相關資訊，目前為止，尚未提出合適之測量方法於細胞生長的同時即時量測三維細胞聚落之生長狀況。In order to make cancer research more similar to physiological state, in vitro cell culture also develops from monolayer culture to 3D culture. The three-dimensional culture system mixes cells with a specific proportion of culture colloid, ie, medium and gel. After mixing, the culture is carried out, and the solid phase is provided by the colloid, so that the cancer cells can form a three-dimensional colony. However, cancer-related research, including the establishment of drug screening platforms and the exploration of pathogenic mechanisms, requires accurate quantification of information on cell growth that has been obtained from cultured cells. So far, no suitable measurement method has been proposed for immediate measurement of cell growth. The growth of three-dimensional cell clusters.
習知常見用於量化三維細胞聚落之方法包含兩種；其一，係將細胞於適當的膠體中培養特定的期間後，再將三維細胞聚落自膠體取出，接著以液態培養基混合後進行細胞計數；其二，係將細胞於適當的膠體中培養特定的期間後，直接將細胞染色後以肉眼觀察，或是拍攝後以影像進行計數。然上述兩種方式皆為終點式(end point)測量，造成研究上諸多限制，例如：進行時間進程(time course)實驗時，將耗費大量時間及耗材，亦難以確保每一組實驗之一致性。此外，細胞染色條件亦影響染色效率及正確性，進一步而言，染色時間過短，或退染時間過長，可能造成訊號太弱而影響影像品質；若染色時間過長，或退染時間過短，亦可能造成偽訊號的現象。由此可知，無論是人工計數或肉眼觀察，均存在主觀判斷之及訊號之差異，無法精準量化。There are two commonly used methods for quantifying three-dimensional cell colonies. One is to take the cells in a suitable colloid for a specific period of time, and then take the three-dimensional cell colonies out of the colloid, and then mix them in a liquid medium for cell counting. Second, after the cells are cultured in a suitable colloid for a specific period of time, the cells are directly stained and visually observed, or imaged for counting. However, both of the above methods are end point measurements, which cause many limitations in research. For example, when performing time course experiments, it takes a lot of time and consumables, and it is difficult to ensure the consistency of each group of experiments. . In addition, the cell staining conditions also affect the dyeing efficiency and correctness. Further, the dyeing time is too short, or the dyeing time is too long, which may cause the signal to be too weak and affect the image quality; if the dyeing time is too long, or the dyeing time is too long Short, it may also cause false signals. It can be seen that there are subjective judgments and signal differences, whether by manual counting or visual observation, which cannot be accurately quantified.
有鑑於上述習知技術之缺點，實有必要建立準確並可即時量測細胞聚落之方法，以期促進癌症研究之效率及精準度。In view of the shortcomings of the above-mentioned prior art, it is necessary to establish an accurate and immediate method for measuring cell colonies in order to promote the efficiency and precision of cancer research.
本發明之一目的，係提供一種即時量測細胞聚落之裝置及其方法，其係利用一導電介質層來調整細胞培養腔室內之細胞培養環境之電阻值，使其得以藉由量測電阻抗值以於細胞生長為細胞聚落之過程中即時獲得細胞生長狀況。An object of the present invention is to provide an apparatus for instantaneous measurement of cell colony and a method thereof, which utilizes a conductive medium layer to adjust the resistance value of a cell culture environment in a cell culture chamber, thereby enabling measurement of electrical impedance The value is obtained immediately in the process of cell growth as a cell colony.
本發明之一目的，係提供一種即時量測細胞聚落之裝置及其方法，其係以一液態培養基將至少一細胞並培養於一導電介質層上，使該細胞得以於該導電介質層表面生長為細胞聚落，並於形成細胞聚落之過程中即時量測電阻抗值以獲得細胞生長狀況。An object of the present invention is to provide an apparatus for instantaneous measurement of cell colony and a method thereof, wherein at least one cell is cultured on a conductive medium layer in a liquid medium to allow the cell to grow on the surface of the conductive medium layer. For cell clustering, and immediately measuring the electrical impedance during the formation of cell colonies to obtain cell growth status.
本發明之一目的，係提供一種即時量測細胞聚落之裝置及其方法，其係以一非液態培養基將至少一細胞培養於一導電介質層上，使該細胞得以懸浮於該非液態培養基中生長為細胞聚落，並於形成細胞聚落之過程中即時量測電阻抗值以獲得細胞生長狀況。An object of the present invention is to provide an apparatus for instantaneous measurement of cell colonies and a method thereof, wherein at least one cell is cultured on a conductive medium layer in a non-liquid medium, and the cells are suspended in the non-liquid medium for growth. For cell clustering, and immediately measuring the electrical impedance during the formation of cell colonies to obtain cell growth status.
為了達到上述之目的，本發明揭示了一種即時量測細胞聚落之方法，其係包含步驟：設置至少一細胞培養腔室於一非導電基板上；形成一導電介質層於該非導電基板上且於該細胞培養腔室內；以一培養基於該導電介質層上培養至少一細胞；提供一電源，使一正極及一負極產生電流導通，並於該導電介質層、該培養基及該細胞形成一電場；以及於該細胞之培養過程中量測一電阻抗值。In order to achieve the above object, the present invention discloses a method for instantaneously measuring cell colonization, which comprises the steps of: providing at least one cell culture chamber on a non-conductive substrate; forming a conductive medium layer on the non-conductive substrate and The cell culture chamber; culturing at least one cell on the conductive medium layer with a medium; providing a power source to cause a positive electrode and a negative electrode to conduct current, and forming an electric field in the conductive medium layer, the medium and the cell; And measuring an electrical impedance value during the cultivation of the cell.
而本發明亦揭示一種用於上述即時量測細胞聚落方法之裝置，其係包含：一非導電基板；至少一細胞培養腔室，其係設置於該非導電基板上；一導電介質層，其係設置於該非導電基板上且於該細胞培養腔室內；以及一正極及一負極，係於一電源提供後產生電流導通；其中，至少一細胞以一培養基培養於該導電介質層上，並於該正極及該負極產生電流導通後，於該導電介質層、該培養基及該細胞形成一電場。The present invention also discloses an apparatus for the above-mentioned instant measurement cell colony method, which comprises: a non-conductive substrate; at least one cell culture chamber disposed on the non-conductive substrate; and a conductive medium layer Provided on the non-conductive substrate and in the cell culture chamber; and a positive electrode and a negative electrode are electrically connected after being supplied by a power source; wherein at least one cell is cultured on the conductive medium layer in a medium, and After the positive electrode and the negative electrode generate a current, an electric field is formed in the conductive medium layer, the medium, and the cell.
本發明之一實施例中，其亦揭露該導電介質層之材料係選自由瓊脂糖凝膠(agarose hydrogel)及甲基纖維素(methylcellulose, MC)所組成之群組中之一或其任意組合。In one embodiment of the present invention, it is also disclosed that the material of the conductive medium layer is selected from one group consisting of agarose hydrogel and methyl cellulose (MC), or any combination thereof. .
本發明之一實施例中，其亦揭露該培養基係一液態培養基或一非液態培養基。In an embodiment of the invention, it is also disclosed that the medium is a liquid medium or a non-liquid medium.
本發明之一實施例中，其亦揭露該細胞係於該導電介質層表面生長為至少一細胞聚落(cell colony)，或懸浮於該非液態培養基中生長為至少一細胞聚落。In one embodiment of the present invention, the cell line is grown on the surface of the conductive medium layer to form at least one cell colony, or suspended in the non-liquid medium to grow into at least one cell colony.
本發明之一實施例中，其亦揭露該正極及該負極係平行設置於該導電介質層下。In an embodiment of the invention, it is also disclosed that the positive electrode and the negative electrode are disposed in parallel under the conductive medium layer.
本發明之一實施例中，其亦揭露該正極及該負極係分別設置於該導電介質層下及該細胞培養腔室上。In an embodiment of the invention, it is also disclosed that the positive electrode and the negative electrode are respectively disposed under the conductive medium layer and on the cell culture chamber.
為使對本發明之特徵及所達成之功效有更進一步之瞭解與認識，謹佐以較佳之實施例及配合詳細之說明，說明如後：For a better understanding and understanding of the features and advantages of the invention, the preferred embodiments and the detailed description are described as follows:
本實施案例提供一種即時量測細胞聚落之裝置及其方法，其目的係解決目前尚未有合適之方法，得以於細胞生長為細胞聚落之過程中，即時獲得細胞生長狀況，導致癌症研究受到限制，且缺乏效率及準確性。利用本實施案例所揭示之方法，可根據實驗需求，即時記錄細胞聚落生長情形，因此實驗設計毋須受到時間、耗材及人力的限制，解決習知僅能終點式(end point)測量之瓶頸。以下，將針對本發明之各實施案例進行說明。The present embodiment provides a device for instantaneous measurement of cell colony and a method thereof, and the object thereof is to solve the problem that the cell growth state is obtained in the process of cell growth and cell colony, and the cancer research is limited. And lack of efficiency and accuracy. Using the method disclosed in this embodiment, the cell colony growth can be recorded in real time according to the experimental requirements. Therefore, the experimental design is not limited by time, consumables and manpower, and the bottleneck of the end point measurement can be solved. Hereinafter, each embodiment of the present invention will be described.
首先，請參閱第1圖、第2A圖及第2B圖，其係本發明之方法流程圖、本發明之第一實施例之裝置示意圖及本發明之第一實施例之電場方向示意圖。如圖所示，第一實施例之方法包含下述步驟：First, please refer to FIG. 1 , FIG. 2A and FIG. 2B , which are a flow chart of the method of the present invention, a schematic diagram of the apparatus of the first embodiment of the present invention, and a schematic diagram of the electric field direction of the first embodiment of the present invention. As shown, the method of the first embodiment comprises the following steps:
步驟S10：設置至少一細胞培養腔室於一非導電基板上；Step S10: setting at least one cell culture chamber on a non-conductive substrate;
步驟S12：形成一導電介質層於該非導電基板上且於該細胞培養腔室內；Step S12: forming a conductive medium layer on the non-conductive substrate and in the cell culture chamber;
步驟S14：以一培養基於該導電介質層上培養至少一細胞；Step S14: culturing at least one cell on the conductive medium layer with a medium;
步驟S16：提供一電源，使一正極及一負極產生電流導通，並於該導電介質層、該培養基及該細胞形成一電場；以及Step S16: providing a power source to cause a positive electrode and a negative electrode to conduct current, and forming an electric field in the conductive medium layer, the medium and the cell;
步驟S18：於該細胞之培養過程中量測一電阻抗值。Step S18: measuring an electrical impedance value during the culture of the cell.
如第2A圖所示，本實施例於步驟S10中，係先提供該非導電基板100，其材料可選用玻璃，但不限於此，該非導電基板100包含該正極102及該負極104，使其得以於供應電源後產生如第2B圖所示之電場；該非導電基板100所包含之電極為平面電極，並可以指叉式排列於該非導電基板100上，但電極之形式不限於此。As shown in FIG. 2A, in the step S10, the non-conductive substrate 100 is first provided, and the material thereof may be glass. However, the non-conductive substrate 100 includes the positive electrode 102 and the negative electrode 104. An electric field as shown in FIG. 2B is generated after the power supply is supplied; the electrode included in the non-conductive substrate 100 is a planar electrode, and may be interdigitated on the non-conductive substrate 100, but the form of the electrode is not limited thereto.
接著，設置該細胞培養腔室110於該非導電基板100，使該細胞培養腔室110位於該正極102及該負極104之間；於本實施例中，可依據實驗需求決定所設置之細胞培養腔室之數量，包含常見之24孔、48孔及96孔之細胞培養腔室，每一細胞培養腔室即代表一個獨立的細胞培養區域。Next, the cell culture chamber 110 is disposed on the non-conductive substrate 100 such that the cell culture chamber 110 is located between the positive electrode 102 and the negative electrode 104. In this embodiment, the set cell culture chamber can be determined according to experimental requirements. The number of chambers contains the usual 24-well, 48-well, and 96-well cell culture chambers, each of which represents an independent cell culture region.
而後，形成該導電介質層120於該非導電基板100上，並位於該細胞培養腔室110之內；所述之該導電介質層之材料係可選用瓊脂糖凝膠(agarose hydrogel)、甲基纖維素(methylcellulose, MC)或兩者之組合，但所選用之材料及使用之比例不在此限。Then, the conductive medium layer 120 is formed on the non-conductive substrate 100 and located in the cell culture chamber 110; the material of the conductive medium layer may be agarose hydrogel or methyl fiber. (methylcellulose, MC) or a combination of the two, but the materials used and the proportions used are not limited.
接續係將預混於該培養基140之該細胞注入該細胞培養腔室110，並於該導電介質層120上，其中，該培養基140係一液態培養基，其係依據所培養之細胞類型選擇；於本實施例中，該細胞將貼附於該導電介質層120之表面，隨著培養時間增加而生長為一細胞聚落130，由於生物細胞具有極低的導電性，當電子移動過程中受到該細胞聚落130阻礙，即可偵測到電阻抗值，電阻抗值越大意即細胞聚落越大，如此即可於形成該細胞聚落130之過程中，即時量測電阻抗值，所獲得之電阻抗值包含電阻抗大小以及電阻抗相角，其分別對應細胞數目及聚落大小，藉此以即時得知細胞的生長狀態。The ligating unit injects the cells premixed with the medium 140 into the cell culture chamber 110 and on the conductive medium layer 120, wherein the medium 140 is a liquid medium, which is selected according to the type of cells to be cultured; In this embodiment, the cell will be attached to the surface of the conductive medium layer 120 and grow into a cell cluster 130 as the culture time increases. Since the biological cell has extremely low conductivity, the cell is exposed to the electron during movement. When the settlement 130 is hindered, the electrical impedance value can be detected. The larger the electrical impedance value is, the larger the cell concentration is, so that the electrical impedance value can be measured in the process of forming the cell cluster 130, and the obtained electrical impedance value is obtained. It includes the electrical impedance and the electrical impedance phase angle, which correspond to the number of cells and the size of the colony, respectively, so as to instantly know the growth state of the cells.
請搭配參閱第1圖、第3A圖及第3B圖，其係本發明之方法流程圖、本發明之第二實施例之裝置示意圖及本發明之第二實施例之電場方向示意圖。如圖所示，本實施例之量測方法包含步驟如下：Please refer to FIG. 1 , FIG. 3A and FIG. 3B , which are a flow chart of the method of the present invention, a schematic diagram of the apparatus of the second embodiment of the present invention, and a schematic diagram of the electric field direction of the second embodiment of the present invention. As shown in the figure, the measurement method of this embodiment includes the following steps:
於本實施例中，僅電極相對於細胞之設置方向，以及細胞生長方式與前述之第一實施例不同，說明如下：In the present embodiment, only the direction in which the electrodes are arranged with respect to the cells, and the manner in which the cells are grown are different from those of the first embodiment described above, and are explained as follows:
如第3A圖所示，本實施例之該電極200，可為正極或負極，且該電極202係對應該電極200為負極或正極，使其得以於供應電源後產生如第3B圖所示之電場。As shown in FIG. 3A, the electrode 200 of the embodiment may be a positive electrode or a negative electrode, and the electrode 202 corresponds to the electrode 200 as a negative electrode or a positive electrode, so that after being supplied with a power source, the electrode 200 is generated as shown in FIG. 3B. electric field.
此外，於本實施例中，該培養基240係一非液態培養基，其係依據所培養之細胞選擇不同種類之培養基，並與各式比例之瓊脂糖凝膠、甲基纖維素或兩者之組合進行混合，所述之比例係依照不同器官之細胞所需之生長環境硬度進行調整，於均勻混合後注入該細胞培養腔室210，並進一步以一液態培養基250將該細胞培養腔室210之空隙填滿，使電子得以流通；於本實施例中，該細胞將懸浮於該培養基240中，並隨著培養時間增加進而生長為一細胞聚落230。In addition, in the present embodiment, the medium 240 is a non-liquid medium, which is selected according to the cultured cells, and is combined with various proportions of agarose gel, methyl cellulose or a combination of the two. The mixing is performed, and the ratio is adjusted according to the hardness of the growth environment required by the cells of different organs, and after uniformly mixing, the cell culture chamber 210 is injected, and the cell culture chamber 210 is further separated by a liquid medium 250. The cells are filled to allow the electrons to circulate; in this embodiment, the cells will be suspended in the medium 240 and grow into a cell colony 230 as the culture time increases.
由於細胞培養基含有數種鹽類及氨基酸等物質，均為良好的導電體，如此當培養基中存在具電阻特性之物質時，將因導電率太高而無法量測以獲得電阻值之變化；據此，本實施例係形成該導電介質層220於該電極200上，藉由上述之導電介質層來提高細胞周圍環境之電阻，；所述之該導電介質層之材料係可選用瓊脂糖凝膠(agarose hydrogel)、甲基纖維素(methylcellulose, MC)或兩者之組合，當兩者組合使用時，其較佳比例為5：5，但所選用之材料及使用之比例不在此限。如此使得本發明之量測方法得以量測因細胞存在培養基中所造成電阻值之變化，達到即時量測細胞聚落之目的。Since the cell culture medium contains several kinds of salts and amino acids, it is a good electrical conductor. When a substance having a resistive property exists in the medium, the conductivity may be too high to be measured to obtain a change in the resistance value; Therefore, in this embodiment, the conductive medium layer 220 is formed on the electrode 200, and the electrical resistance of the surrounding environment of the cell is improved by the conductive medium layer; the material of the conductive medium layer is selected from agarose gel. (agarose hydrogel), methylcellulose (MC), or a combination of the two, when the two are used in combination, the preferred ratio is 5:5, but the materials selected and the proportions used are not limited thereto. Thus, the measuring method of the present invention can measure the change of the electric resistance value caused by the presence of the cells in the medium, and achieve the purpose of measuring the cell colony in an instant.
以下例舉特定實施例，然其僅為例示說明，而非用以限制本發明其他形式實施。The following examples are intended to be illustrative, and are not intended to limit the invention.
[實施例1][Example 1]
細胞培養將人類肝癌細胞(Human hepatoma cells, Huh7)以改良杜氏伊格爾培養基(Dulbecco’s modified eagle medium, DMEM)培養於培養盤中，該培養基係包含10%之胎牛血清、100 U/mL 之青黴素鈉(penicillin G sodium)、100 mg/mL 之鏈黴素(streptomycin)及d 0.25 mg/mL之兩性黴素B(amphotericin B)。該人類肝癌細胞係以標準細胞培養流程放大其細胞數，待所生長之細胞數足以進行實驗時，再以0.05%之胰蛋白酶(trypsin)水解細胞貼附於培養盤之結構，使得細胞脫離培養盤，計數細胞並進行後續流程。Cell culture Human hepatoma cells (Huh7) were cultured in a culture dish in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum, 100 U/mL. Penicillin G sodium, 100 mg/mL streptomycin and d 0.25 mg/mL amphotericin B (amphotericin B). The human hepatoma cell line amplifies the number of cells in a standard cell culture process. When the number of cells to be grown is sufficient for the experiment, the cells are attached to the structure of the culture plate by 0.05% trypsin, so that the cells are detached from the culture. Plate, count cells and follow up the process.
製備可即時量測電阻抗之細胞培養裝置提供一個包含10個細胞培養腔室之細胞培養裝置，該細胞培養裝置係包含一玻璃基板，其嵌有10個鉻/金平面指叉式電極(planar interdigitated electrodes)甲、一聚二甲基矽氧烷(polydimethylsiloxane)層，並透過一元件將其區隔成十個培養區域，並對應於該玻璃基板之該些電極。於執行細胞培養前，該細胞培養裝置需經過清洗、殺菌並於紫外光下照射至少16個小時。A cell culture device forpreparing an electrical impedance measuring device provides acell culture device comprising 10 cell culture chambers, the cell culture device comprising a glass substrate embedded with 10 chromium/gold planar interdigitated electrodes (planar) Interdigitated electrode A layer of polydimethylsiloxane, which is divided into ten culture regions by an element and corresponding to the electrodes of the glass substrate. Prior to performing cell culture, the cell culture device is cleaned, sterilized, and irradiated under ultraviolet light for at least 16 hours.
形成導電介質層將1% (w/v)之瓊脂糖凝膠及1% (w/v)之甲基纖維素以5：5之比例均勻混合，並注入各細胞培養腔室中，待凝固後即於該玻璃基板上形成導電介質層。Forming a conductive medium layer 1% (w/v) agarose gel and 1% (w/v) methylcellulose are uniformly mixed at a ratio of 5:5, and injected into each cell culture chamber to be coagulated Thereafter, a conductive medium layer is formed on the glass substrate.
細胞聚落之培養於前述之細胞培養步驟中所得之細胞中取得10000顆，並與前述之培養基均勻混合，置於上述之細胞培養裝置中，使其於導電介質層之表面生長，並於37°C、5%二氧化碳及溼度適當之培養箱中培養7天。Cell culture is obtained by culturing 10000 cells in the cell culture step described above, and uniformly mixed with the aforementioned medium, placed in the above cell culture device, and grown on the surface of the conductive medium layer at 37°. C, 5% carbon dioxide and humidity in an appropriate incubator for 7 days.
藥物處理上述之細胞經過三天的培養後即形成細胞聚落，接著製備濃度不同之抗癌藥物阿黴素(Doxorubicin)並以DMEM培養基調整其體積至20 L，於第60個小時，即第2.5天時，分別將不同濃度之阿黴素加到不同的培養區域中，且每24小時添加一次抗癌藥物。Drug treatment of the above cells after three days of culture to form cell colonies, followed by the preparation of different concentrations of the anticancer drug Doxorubicin (Doxorubicin) and adjusted its volume to 20 L in DMEM medium, at the 60th hour, ie 2.5 At different times, different concentrations of doxorubicin were added to different culture areas, and anticancer drugs were added every 24 hours.
於細胞聚落形成之過程中即時量測電阻抗 提供0.1有效電壓值(Vrms)之電位至該細胞培養裝置，且以10赫茲(Hz)至100千赫茲(kHz)之條件量測電阻抗值。於本實施例中，係以1千赫茲進行電阻抗之量測，電阻抗之變化可反映細胞聚落之增生數量。結果如第5圖所示。Immediate measurement of electrical impedance during cell formation The potential of 0.1 effective voltage (Vrms) is supplied to the cell culture device, and the electrical impedance value is measured at a condition of 10 Hz to 100 kHz. In the present embodiment, the electrical impedance is measured at 1 kHz, and the change in electrical impedance reflects the amount of proliferation of cell colonies. The result is shown in Figure 5.
第4圖為細胞聚落培養過程中所量測之電阻抗值。如圖所示，在未處理抗癌藥物之前，細胞數量均隨著培養時間增加而增加，於培養第2.5天時對細胞聚落處理抗癌藥物，處理抗癌藥物之細胞與未處理抗癌藥物之細胞相較之下，其細胞數量隨著培養時間增加而逐漸減少，且細胞存活數量與抗癌藥物之濃度呈現負相關的趨勢。如此可見，本實施例所提供之量測方法，確實能夠精準並正確地量測細胞數量，清楚顯示抗癌藥物之毒殺效果，具有高度靈敏性。Figure 4 shows the electrical impedance values measured during cell colony culture. As shown in the figure, before the anticancer drug was treated, the number of cells increased with the increase of the culture time. On the 2.5th day of culture, the anticancer drug was treated for cell colonization, and the anticancer drug cells and untreated anticancer drugs were treated. In contrast, the number of cells gradually decreased with the increase of culture time, and the number of cell survival showed a negative correlation with the concentration of anticancer drugs. It can be seen that the measurement method provided by the embodiment can accurately measure the number of cells accurately and correctly, clearly show the poisoning effect of the anticancer drug, and has high sensitivity.
[實施例2][Embodiment 2]
細胞培養與實施例1所述之方法相同，如此不再贅述。The cell culture was the same as that described in Example 1, and thus will not be described again.
製備可即時量測電阻抗之細胞培養裝置提供一包含9個細胞培養腔室之細胞培養裝置，該細胞培養裝置係包含一玻璃基板，其嵌有9個鉻/金量測電極、一聚二甲基矽氧烷(polydimethylsiloxane)層，並透過一元件將其區隔成九個培養區域，並對應於該玻璃基板之該些量測電極以及一ITO導電層，其作為接地電極，如此，當提供電源時即可於導電介質層、培養基及細胞聚落形成電場，藉此以量測電阻抗。於執行細胞培養前，該細胞培養裝置需經過清洗、殺菌並於紫外光下照射至少16個小時。A cell culture device forpreparing an electrical impedance measuring device provides acell culture device comprising nine cell culture chambers, the cell culture device comprising a glass substrate embedded with nine chromium/gold measuring electrodes, one poly a layer of polydimethylsiloxane, which is partitioned into nine culture regions by an element, and corresponds to the measurement electrodes of the glass substrate and an ITO conductive layer, which serves as a ground electrode, thus When the power source is supplied, an electric field can be formed in the conductive medium layer, the culture medium, and the cell cluster, thereby measuring the electrical impedance. Prior to performing cell culture, the cell culture device is cleaned, sterilized, and irradiated under ultraviolet light for at least 16 hours.
形成導電介質層與實施例1所述之方法相同，如此不再贅述。The formation of the conductive medium layer is the same as that described in Embodiment 1, and will not be described again.
細胞聚落之培養取1% (w/v)之瓊脂糖凝膠(agarose hydrogel)及1.8% (w/v)之甲基纖維素(methylcellulose, MC)以特定比例混合成培養膠體，於前述之細胞培養步驟中所得之細胞中取得10000顆，並以1.8% (w/v)之甲基纖維素混合並包覆該些細胞，從中取得濃度為50 L cells/MC之細胞混合液，並置於上述之細胞培養裝置中，接著再將前述之培養膠體置於該細胞混合液上，於37°C、5%二氧化碳及溼度適當之培養箱中培養7天。Cell colony culture 1% (w/v) agarose hydrogel and 1.8% (w/v) methylcellulose (MC) were mixed into a culture gel in a specific ratio, as described above. 10,000 cells were obtained from the cells in the cell culture step, and the cells were mixed and coated with 1.8% (w/v) of methylcellulose, and a cell mixture of 50 L cells/MC was taken therefrom and placed. In the above cell culture apparatus, the culture gel described above was placed on the cell mixture, and cultured in an incubator at 37 ° C, 5% carbon dioxide, and humidity for 7 days.
藥物處理製備濃度不同之抗癌藥物阿黴素(Doxorubicin)並以DMEM培養基調整其體積至100 L，於細胞經過三天的培養後，分別將不同濃度之阿黴素加到不同的細胞培養腔室中。所述之阿黴素係一種常見的抗癌藥物，本實施例利用阿黴素確認本發明之量測方法用於即時量測細胞聚落時之實用性及靈敏度。Drug treatment to prepare Doxorubicin, a different concentration of the anticancer drug, and adjust its volume to 100 L in DMEM medium. After three days of culture, different concentrations of doxorubicin were added to different cell culture chambers. In the room. The doxorubicin is a common anticancer drug, and the present embodiment uses doxorubicin to confirm the utility and sensitivity of the measuring method of the present invention for instantaneous measurement of cell colonies.
於細胞聚落形成之過程中即時量測電阻抗於細胞聚落培養期間，可依據實驗需求隨時量測電阻抗。提供0.1有效電壓值(Vrms)之電位至該細胞培養裝置，且以10赫茲(Hz)至100千赫茲(kHz)之條件量測電阻抗值。於本實施例中，係以1千赫茲進行電阻抗之量測，所獲得之電阻抗大小之變化可反映細胞聚落之增生數量，所獲得之電阻抗相角(impedance phase angle)則可反映細胞聚落之大小。結果如第6A圖、第6B及第6C圖所示。Immediate measurement of electrical impedance during cell colony formation During cell colony culture,electrical impedance can be measured at any time according to experimental needs. A potential of 0.1 effective voltage value (Vrms) is supplied to the cell culture device, and the electrical impedance value is measured at a condition of 10 Hz to 100 kHz. In the present embodiment, the electrical impedance is measured at 1 kHz, and the change in the magnitude of the electrical impedance obtained reflects the proliferation of cell colonies, and the obtained impedance phase angle reflects the cells. The size of the settlement. The results are shown in Figures 6A, 6B and 6C.
第5A圖係以顯微鏡拍攝之細胞聚落圖，其拍攝之時間點分別為細胞培養後第1、第3、第5及第7天；第5B圖係於細胞聚落培養過程中所量測之電阻抗值；第5C圖係於細胞聚落培養過程中所量測之電阻抗相角。如圖所示，在未處理抗癌藥物之前，細胞數量均隨著培養時間增加而增加，且細胞聚落大小亦隨著培養時間增加而增大，於培養第三天時對細胞聚落處理抗癌藥物，處理抗癌藥物之細胞與未處理抗癌藥物之細胞相較之下，其細胞數量隨著培養時間增加而逐漸減少，且細胞存活數量與抗癌藥物之濃度呈現負相關的趨勢，此外，細胞聚落之大小亦隨著培養時間增加而逐漸變小，並同樣與抗癌藥物之濃度呈現負相關的趨勢。由此可以，本實施例所提供之量測方法，確實能夠精準並正確地量測細胞聚落之細胞數量及細胞聚落之大小，亦具有高度靈敏性。Fig. 5A is a cell colony image taken by a microscope, and the time points of the photographing are the first, third, fifth, and seventh days after cell culture, respectively; and the fifth panel is the electricity measured during the cell colony culture. Impedance value; Figure 5C is the electrical impedance phase angle measured during cell colony culture. As shown in the figure, before the anticancer drug was treated, the number of cells increased with the increase of the culture time, and the cell colony size also increased with the increase of the culture time, and the cell colony was treated for anti-cancer on the third day of culture. Compared with cells with untreated anticancer drugs, the number of cells treated with anticancer drugs gradually decreased with the increase of culture time, and the number of cell survival showed a negative correlation with the concentration of anticancer drugs. The size of cell colonies also gradually decreased with the increase of culture time, and also showed a negative correlation with the concentration of anticancer drugs. Therefore, the measurement method provided by the embodiment can accurately and accurately measure the number of cells and the size of cell colonies of the cell colony, and is also highly sensitive.
綜上所述，本發明所提供之即時量測細胞聚落之裝置及其方法，其利用導電介質層調整細胞培養腔室內之細胞培養環境之電阻抗，使其得以於導電介質層、培養基及細胞聚落產生電場，如此可經量測之後獲得電阻抗值及電阻抗相角，再透過準確量化前述之資訊而獲得細胞聚落之細胞數量及細胞聚落大小等細胞生長相關資訊，解決習知以人工計算細胞數或以影像計算細胞數之精準度及效率不佳等問題。In summary, the present invention provides an apparatus for instantaneous measurement of cell clustering and a method thereof, which utilizes a conductive medium layer to adjust an electrical impedance of a cell culture environment in a cell culture chamber to enable a conductive medium layer, a medium, and a cell. The cluster generates an electric field, so that the electrical impedance value and the electrical impedance phase angle can be obtained after measurement, and then the cell growth related information such as the number of cell colonies and the cell colony size can be obtained by accurately quantifying the above information, and the conventional calculation is manually calculated. The number of cells or the accuracy and efficiency of calculating the number of cells by imaging.
惟以上所述者，僅為本發明之較佳實施例而已，並非用來限定本發明實施之範圍，舉凡依本發明申請專利範圍所述之形狀、構造、特徵及精神所為之均等變化與修飾，均應包括於本發明之申請專利範圍內。The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and the variations, modifications, and modifications of the shapes, structures, features, and spirits described in the claims of the present invention. All should be included in the scope of the patent application of the present invention.
100‧‧‧非導電基板100‧‧‧ Non-conductive substrate
102‧‧‧正極102‧‧‧ positive
104‧‧‧負極104‧‧‧negative
110‧‧‧細胞培養腔室110‧‧‧ cell culture chamber
120‧‧‧導電介質層120‧‧‧ Conductive dielectric layer
130‧‧‧細胞聚落130‧‧‧cell colony
140‧‧‧培養基140‧‧‧ medium
200‧‧‧電極200‧‧‧electrode
202‧‧‧電極202‧‧‧electrode
210‧‧‧細胞培養腔室210‧‧‧ cell culture chamber
220‧‧‧導電介質層220‧‧‧ Conductive dielectric layer
230‧‧‧細胞聚落230‧‧‧cell settlement
240‧‧‧培養基240‧‧‧ medium
250‧‧‧液態培養基250‧‧‧Liquid medium
第1圖：其係本發明之方法流程圖； 第2A圖：其係本發明之第一實施例之裝置示意圖； 第2B圖：其係本發明之第一實施例之電場方向示意圖； 第3A圖：其係本發明之第二實施例之裝置示意圖； 第3B圖：其係本發明之第二實施例之電場方向示意圖； 第4圖：其係本發明之第一實施例之細胞聚落培養過程中所量測之電阻抗值； 第5A圖：其係本發明之第二實施例之顯微鏡拍攝之細胞聚落圖； 第5B圖：其係本發明之第二實施例之細胞聚落培養過程中所量測之電阻抗值；以及 第5C圖：其係本發明之第二實施例之細胞聚落培養過程中所量測之電阻抗相角。1 is a flow chart of a method of the present invention; FIG. 2A is a schematic view of a device according to a first embodiment of the present invention; FIG. 2B is a schematic view showing a direction of an electric field according to a first embodiment of the present invention; Figure: is a schematic view of a device according to a second embodiment of the present invention; Figure 3B is a schematic view of the electric field direction of the second embodiment of the present invention; Figure 4 is a cell colony culture of the first embodiment of the present invention The electrical impedance value measured in the process; FIG. 5A is a microscopic photograph of the cell colony of the second embodiment of the present invention; FIG. 5B is a cell colony culture process of the second embodiment of the present invention The measured electrical impedance value; and Figure 5C: which is the electrical impedance phase angle measured during the cell colony culture process of the second embodiment of the present invention.
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| TW106106898ATWI680295B (en) | 2017-03-02 | 2017-03-02 | Device and method for measuring cell colony in real time |
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| TW106106898ATWI680295B (en) | 2017-03-02 | 2017-03-02 | Device and method for measuring cell colony in real time |
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| Date | Code | Title | Description |
|---|---|---|---|
| MM4A | Annulment or lapse of patent due to non-payment of fees |