WO2011043077A1 - 未分化多能性幹細胞の識別方法及び装置並びに自動培養方法及び装置 - Google Patents
未分化多能性幹細胞の識別方法及び装置並びに自動培養方法及び装置 Download PDFInfo
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- the present invention relates to an identification method and apparatus for undifferentiated pluripotent stem cells and an automatic culture method and apparatus, and more specifically, a method and apparatus for identifying undifferentiated pluripotent stem cells and other pluripotent stem cells. And an automatic culture method and apparatus for undifferentiated pluripotent stem cells using these methods and apparatuses.
- pluripotent stem cells such as ES cells and iPS cells (in this specification, “ES cells” and “iPS cells” are collectively referred to as “pluripotent stem cells”) have been artificially created. Therefore, a great contribution is expected in fields such as regenerative medicine. Since pluripotent stem cells have the pluripotency that can be differentiated into various cells constituting the living body, by using the patient's own iPS cells, skin, cartilage, bone, blood vessels, It becomes possible to regenerate nerves, organs and the like.
- pluripotent stem cells have pluripotency as described above, some pluripotent stem cells may start to differentiate during the culture. Thus, a cell that has started differentiation cannot be returned to an undifferentiated state again, and cannot be used to create a target organ or organ. Therefore, in the passage of pluripotent stem cells, it is important to selectively pass only undifferentiated pluripotent stem cells.
- Such identification of undifferentiated pluripotent stem cells can be performed, for example, by using “CellSelector” of “AVISO” by staining cells or observing fluorescence.
- staining is often performed after the cells are fixed, and since the dye is often toxic to the cells, it is difficult to observe the cells alive. Even if a low-toxic dye is used, it is still toxic to cells and inappropriate for application in the field of regenerative medicine.
- the present invention has been made to solve the above-mentioned problems of the prior art, and the object of the present invention is to achieve undifferentiated multiplicity even when some of the pluripotent stem cells start to differentiate during culture. It is to provide a method and apparatus capable of selectively identifying colonies consisting only of pluripotent stem cells, and to provide a method and apparatus capable of automatically culturing only undifferentiated pluripotent stem cells.
- the colony identification method of the present invention is a method for identifying a colony based on a captured image of a colony composed of pluripotent stem cells in a culture vessel, wherein the differentiated pluripotent stem cell is based on the luminance in the captured image. And a differentiated colony containing only undifferentiated pluripotent stem cells.
- pluripotent stem cells can no longer be said to have “pluripotency”, but in the present application, this term will be used for convenience.
- a multilayer colony containing pluripotent stem cells stacked in multiple layers can be further identified based on the brightness of the colony.
- a colony having an area brighter than the first threshold value of the brightness is determined to be the differentiated colony, and a colony having only an area having a brightness equal to or lower than the first threshold value is not determined. It can be configured to determine that it is a differentiated colony.
- a colony having a brighter area than the first threshold value of the luminance is determined to be the differentiated colony, and has a luminance equal to or lower than the first threshold value and equal to or brighter than the second threshold value.
- a colony having only a region may be determined as the undifferentiated colony, and a colony having a region with a luminance lower than the second threshold may be determined as the multilayer colony.
- the first threshold value is a discriminant analysis with respect to the distribution of the number of pixels for each luminance value obtained for a median filter image obtained by subjecting the captured image to processing by one or more median filters. Required by applying the law.
- the second threshold value can be obtained as follows. That is, among the pixels of the smoothed filter image obtained by subjecting the captured image to one or more times of smoothing filter processing, the first threshold value is multiplied by a predetermined magnification in the range of 105 to 115%.
- the method for automatically culturing undifferentiated pluripotent stem cells of the present invention comprises a step of discriminating the undifferentiated colony from colonies other than the undifferentiated colony by the identification method described above, the undifferentiated colony and the undifferentiated colony. Obtained by obtaining position information of colonies other than differentiated colonies, introducing a cell detaching agent into the culture vessel, detaching the undifferentiated colonies based on the position information, and detaching the undifferentiated colonies. A step of recovering the undifferentiated pluripotent stem cells obtained.
- the method for automatically culturing undifferentiated pluripotent stem cells of the present invention comprises the step of discriminating between the undifferentiated colony and a colony other than the undifferentiated colony by the colony identifying method according to any one of the above, Obtaining colony and location information of colonies other than the undifferentiated colony, introducing a cell detaching agent into the culture container, detaching colonies other than the undifferentiated colonies based on the location information, It includes a step of discarding pluripotent stem cells obtained by exfoliating colonies other than differentiated colonies, and a step of recovering undifferentiated pluripotent stem cells by exfoliating the undifferentiated colonies.
- the colony identification apparatus includes an image input unit that inputs a captured image that has been image-processed based on a luminance value, and a differentiated colony that includes a differentiated pluripotent stem cell based on the luminance of each colony. And an identification means for distinguishing undifferentiated colonies consisting only of pluripotent stem cells.
- the identification means can be configured to further identify a multi-layered colony including multi-layered pluripotent stem cells based on the brightness of the colony.
- the identification unit determines that a colony having a brightness area brighter than the first threshold value of the brightness is the differentiated colony and has only a brightness area equal to or lower than the first threshold value.
- a colony can be configured to determine that it is the undifferentiated colony.
- the identification unit determines that a colony having an area brighter than the first threshold value of the luminance is the differentiated colony, and is equal to the first threshold value or darker than the second threshold value? It is also possible to determine that a colony having only a brighter area than this is an undifferentiated colony, and to determine that a colony having a darker area than the second threshold is the multilayer colony. It is.
- the first threshold value is a discriminant analysis with respect to the distribution of the number of pixels for each luminance value obtained for a median filter image obtained by subjecting the captured image to processing by one or more median filters. Required by applying the law.
- the second threshold value can be obtained as follows. That is, among the pixels of the smoothed filter image obtained by subjecting the captured image to one or more times of smoothing filter processing, the first threshold value is multiplied by a predetermined magnification in the range of 105 to 115%.
- An automatic culture apparatus for undifferentiated pluripotent stem cells includes a colony identification device according to any one of the above, a release agent introduction means for introducing a cell release agent into the culture container, and position information of each colony. And a pipetting device for removing undifferentiated colonies and collecting undifferentiated pluripotent stem cells obtained by exfoliating the undifferentiated colonies.
- the automatic culture apparatus for undifferentiated pluripotent stem cells of the present invention includes the colony identification apparatus, position information acquisition means for acquiring position information of the undifferentiated colonies and colonies other than the undifferentiated colonies, and the culture Based on the location information acquired by the release agent introduction means for introducing the cell release agent into the container and the location information acquisition means, the colonies other than the undifferentiated colonies are exfoliated and obtained by exfoliating the colonies other than the undifferentiated colonies. And a pipetting device that discards the pluripotent stem cells obtained and further detaches the undifferentiated colonies to recover the undifferentiated pluripotent stem cells.
- the colony identification method and apparatus of the present invention obtains a captured image of a colony composed of pluripotent stem cells in a culture vessel, and performs image processing on this to differentiate differentiated and undifferentiated colonies based on the brightness of each colony. It is possible to identify. Therefore, even when some of the colonies start to differentiate during culturing of pluripotent stem cells, only undifferentiated pluripotent stem cells can be identified. In addition, by identifying colonies in this way, an automatic culture method and apparatus that can selectively pass through only undifferentiated pluripotent stem cells are provided.
- FIG. 5 It is a figure which shows the image which image
- the imaging device camera
- FIG. 19 is a histogram showing a distribution of the number of pixels with respect to a luminance value obtained for the image shown in FIG. 18.
- FIG. It is an image after performing median filter processing 10 times with respect to the image of FIG. It is an image after performing the Gaussian filter process 10 times with respect to the image of FIG. FIG.
- FIG. 22 is a histogram showing a distribution of the number of pixels with respect to a luminance value obtained for the image of FIG. 21.
- FIG. It is a colony extraction image obtained by using a threshold value obtained by multiplying the first threshold value by a predetermined magnification in a range of 105 to 115%. It is a histogram showing distribution of the number of pixels with respect to the luminance value calculated
- the image is composed only of a region having a luminance equal to or darker than the first threshold value 132 and equal to or brighter than the second threshold value 73.
- the image is composed only of a region having a luminance equal to or darker than the first threshold value 140 and equal to or brighter than the second threshold value 73.
- the colony identification method of the present invention differentiation between differentiated colonies containing differentiated pluripotent stem cells and undifferentiated colonies containing only undifferentiated pluripotent stem cells is performed, and pluripotent stem cells stacked in multiple layers. Multi-layer colonies containing can also be identified.
- This identification is performed based on the brightness of each colony in the image after image processing by performing image processing of pluripotent stem cells forming colonies cultured in the culture vessel.
- the image processing mainly means normalizing the luminance of each pixel to a luminance in a range of, for example, monochrome 0 to 255 gradations (8 bits). In this embodiment, the 8-bit 256 gradation is used.
- the present invention is not limited to this, and the number of gradations can be increased or decreased.
- the identification of colonies by luminance is based on the following knowledge. That is, as shown in FIG. 1, when cells or the like are not attached to the bottom surface 10 of the dish, the illumination light 12 passes through the bottom surface 10 of the dish and is hardly scattered and then is imaged by a camera or the like as the imaging light 13. Since it reaches the device 11, the resulting image is bright. On the other hand, as shown in FIG. 2, when pluripotent stem cells or the like are attached to the bottom surface 10 of the dish, the illumination light 12 is scattered by the pluripotent stem cells 21a.
- the proportion of the portion other than the nucleus 22 is small compared to the differentiated pluripotent stem cell described later, and it has a relatively large nucleus 22.
- the illumination light 12 is relatively scattered. Therefore, the imaging light 14 reaching the imaging device 11 is reduced, and the obtained image of the portion of the undifferentiated pluripotent stem cell 21a becomes dark.
- the differentiated pluripotent stem cell has a portion other than the nucleus 22 larger than the nucleus 22, and the portion of the nucleus 22 becomes relatively smaller.
- the scattering received by the illumination light 12 is smaller than in the case of the undifferentiated pluripotent stem cell of FIG. 2, and the imaging light 15 reaching the imaging device 11 is increased. Therefore, the obtained image of the portion of the differentiated pluripotent stem cell 21b becomes brighter than in the case of the undifferentiated pluripotent stem cell of FIG. Further, as shown in FIG. 4, when the pluripotent stem cells are excessively cultured, the illumination light 12 is scattered more than in the case of FIG. Therefore, the imaging light 16 reaching the imaging device 11 becomes very small, and the image of the obtained undifferentiated pluripotent stem cell 21c portion becomes very dark.
- FIG. 5 is an image of a dish obtained by culturing iPS cells, which are one type of pluripotent stem cells.
- FIG. 6 shows an image obtained by processing an image obtained by photographing the region C in FIG. 5 with a phase contrast microscope. This region C is an undifferentiated colony composed of undifferentiated pluripotent stem cells.
- FIG. 7 shows an image obtained by processing an image obtained by photographing the region D in FIG. 5 with a phase contrast microscope. Region E in FIG. 7 is composed of differentiated pluripotent stem cells, and region F is a region where pluripotent stem cells stacked in multiple layers are present. As can be seen from FIG. 6 and FIG.
- the region C of undifferentiated colonies consisting of undifferentiated pluripotent stem cells, the region E of differentiated pluripotent stem cells, and the region F of pluripotent stem cells stacked in multiple layers are: It can be seen that the image can be identified by the brightness of the captured image.
- feeder cells for preparing an iPS cell growth environment may be grown on the entire surface of the dish prior to iPS cell culture, but the feeder cells are considerably smaller than iPS cells. Therefore, it appears as background noise in the captured image.
- FIG. 8 schematically shows a criterion for determining which of the above three areas a specific colony corresponds to.
- the brightness of the colony image is brighter than the first threshold A, it is determined to be a differentiated colony, equal to or darker than the first threshold A, and
- the threshold value B of 2 When it is equal to or brighter than the threshold value B of 2, it is determined to be an undifferentiated region, and a colony having a luminance lower than the second threshold value B is determined to be a region where pluripotent stem cells overlap each other.
- a method for determining the first threshold A will be described.
- a wide-area captured image is taken by a camera 43 through a lid 42 in a dish 41 in which pluripotent stem cells are cultured.
- the image shown in FIG. 18 is obtained by normalizing this image to 256 gradations. Note that the photographing in the dish 41 may be performed with the lid 42 removed.
- a median filter image is created by performing processing by the median filter once or a plurality of times on the image of FIG.
- the median filter has a function of removing images such as feeder cells and fine noise while leaving edge information in the image.
- the number of processings by the median filter is determined according to the magnitude of noise, the actual distance of one pixel in the image, and the filter size.
- the specific explanation when determining the number of times of processing by the median filter is as follows.
- the feeder cells are noisy.
- the size of a general feeder cell is about 300 ⁇ m in length and about 30 ⁇ m in thickness, but there is also a feeder cell having a thickness of 100 ⁇ m in a large one.
- the image shown in FIG. 18 is captured using a camera with an image sensor interval of 4.65 ⁇ m and a lens with a magnification of 0.218 times. In this case, the actual distance of one pixel is 21.3 ⁇ m.
- a filter that performs processing in the range of 3 ⁇ 3 pixels is adopted, and therefore processing is performed using information on the range of the actual distance corresponding to two pixels in the horizontal direction, for example, by one filtering process. .
- processing is performed using information on the range of the actual distance corresponding to 20 pixels.
- the filter process is performed on noise having a size of 100 ⁇ m, which is the maximum thickness of the feeder cell, it is effective to perform the filter process in a range not less than twice the size of the noise.
- the number of times of the filtering process is doubled to 10 times.
- FIG. 20 is an image after the median filter processing is performed 10 times on the image of FIG. From FIG. 20, it can be seen that in the image after the median filter processing, an image or noise that seems to be due to a small feeder cell is removed.
- the distribution of the number of pixels with respect to the luminance value is obtained for the median filter image.
- a method for determining the second threshold B When determining the second threshold value B, first, a colony threshold value for extracting only a region where colonies exist is obtained.
- the colony threshold is determined using a histogram (FIG. 19) representing the distribution of the number of pixels with respect to the luminance value for the image shown in FIG.
- a Gaussian filter image is created by subjecting the image of FIG. 18 to processing by a Gaussian filter which is a kind of a smoothing filter once or a plurality of times.
- the Gaussian filter has a function of removing noise and a function of preventing a significant change in luminance value between the pixel to be processed and the peripheral pixels, thereby obtaining a smooth image.
- FIG. 21 shows an image after the Gaussian filter is applied 10 times
- FIG. 22 is a histogram thereof. From comparison between FIG. 18 and FIG. 21, it can be seen that the image after the Gaussian filter processing is smoother. Further, from comparison between FIG. 19 and FIG. 22, it can be seen that the peak of the histogram is increased and the peak position is also moved after the Gaussian filter processing.
- a colony threshold value for identifying a region where colonies exist and a region where colonies do not exist is obtained.
- the colony threshold value is obtained by multiplying the first threshold value by a predetermined magnification in the range of 105 to 115%. By extracting only pixels having a luminance value smaller than the colony threshold value from the image after the Gaussian filter processing, an image of only the colony region is obtained.
- the magnification to be multiplied by the first threshold is determined by an experiment described below. That is, a temporary colony threshold value is set according to the temporarily set magnification, and a temporary colony extraction image is created using this threshold value.
- the colony extraction range obtained here is compared with the colony range determined by a human through visual observation, microscopic observation, or a stained image. If the colony range determined by a human is large, the magnification is increased. Further, the colony extraction range obtained here is compared with a colony range determined by a human through visual observation, microscopic observation, or a stained image. If the colony range determined by a human is small, the magnification is decreased.
- This multiplication factor is constant if the image capturing conditions, the pluripotent stem cell culture conditions, and the type of pluripotent stem cell line to be cultured are determined.
- the magnification is 110%
- FIG. 23 is a colony extraction image obtained in this way.
- small-area colonies that are judged to be unsuitable for separation and recovery are excluded.
- a region where the luminance value existing inside the colony is larger than 145 is also part of the colony.
- FIG. 24 is a histogram showing the distribution of the number of pixels with respect to the luminance value in FIG. In FIG. 23, all parts other than the colony are white (luminance value 255), so the number of pixels corresponding to the luminance value 255 in FIG. 24 is very large (not shown).
- the second threshold value is obtained.
- the maximum luminance value in the setting. In the present embodiment, the maximum luminance value 130.
- the number of pixels from 90% of the number of pixels (ordinate value) on the histogram corresponding to the maximum luminance value to 20%.
- a straight line is obtained by the method of least squares using the coordinates of the points on the histogram existing until.
- the intersection of the straight line and the horizontal axis obtained by the least square method is obtained as the second threshold value B.
- the second threshold value B 73.
- FIG. 26 is an image of only undifferentiated cells obtained as described above in the present embodiment.
- the luminance value 132 obtained by the discriminant analysis method as described above when it is actually determined to be a differentiated cell even though it is an undifferentiated cell, the luminance value obtained by multiplying the threshold obtained by the discriminant analysis method by a predetermined magnification within a range of 100% to 115% (but not exceeding the magnification for obtaining the colony threshold described above) is the first threshold A. In some cases, undifferentiated cells can be more accurately identified.
- the magnification by which the threshold value obtained by the discriminant analysis method is multiplied is determined by experiment.
- a temporary first threshold is set with a magnification of 100%, and an image of only undifferentiated colonies is created using this threshold.
- the magnification is multiplied by the threshold value obtained by the discriminant analysis method.
- the threshold value calculated using this multiplication factor is set as the first threshold value. This multiplication factor is constant if the image capturing conditions, the pluripotent stem cell culture conditions, and the type of pluripotent stem cell line to be cultured are determined.
- FIG. 9 schematically shows a captured image of a culture container in which undifferentiated colonies and differentiated colonies are mixed. The undifferentiated region is dark and the differentiated portion is shown in white.
- isolated colonies 31 and 35 consisting only of undifferentiated pluripotent stem cells are selected as colonies to be detached and recovered. Colonies 32 whose periphery has started to be differentiated are excluded from colonies to be detached. In addition, colonies 33 that have differentiated as a whole are also excluded. Furthermore, the colony 34 in which the inside is partially differentiated is also excluded from the colonies to be detached.
- the colony 36 is composed only of undifferentiated pluripotent stem cells.
- pluripotent stem cells are collected using a pipetting device.
- a cell peeling agent is introduced into the entire culture container. This stripper can detach cells adhering to the bottom of the dish for a predetermined period of time by the flow of the culture medium by discharging the pipetting device, and the cells are not dished in the absence of liquid flow. The type, concentration, amount, etc. are determined so as not to peel from the bottom surface. After introducing the detachment solution containing the cell detachment agent, as shown in FIG.
- the medium is discharged from the pipetting device 44 to the selected undifferentiated colonies, and only undifferentiated pluripotent stem cells are generated by the solution flow. Will peel off.
- the discharge of the culture medium from the pipetting device is not limited to one time, and can be performed a plurality of times at the same position or while moving, and the discharge speed, the liquid amount, etc. can be changed.
- the dish 41 is tilted as necessary, and the liquid containing the undifferentiated pluripotent stem cells is collected by the pipetting device 44 and further subcultured. When the cost is required, it is dispensed to the dish 41 containing a new medium.
- the upper half of the undifferentiated colony 45 is peeled off by pipetting twice, and after confirming the state, undifferentiated by further pipetting twice.
- the lower half of the colony 45 is peeled off to complete the peeling of the entire colony.
- FIG. 17 when a differentiated colony 46 or the like is present in the vicinity of the undifferentiated colony 45, the region 48 a, the region 48, and the region 48 are not separated without pipetting. Only the three regions 48b and 48c are peeled off by pipetting.
Abstract
Description
11 撮像装置
12 照明光
13,14,15,16 撮像光
21a 未分化の多能性幹細胞
21b 分化した多能性幹細胞
21c 多層に重なった多能性幹細胞
22 核
41 ディッシュ
42 蓋
43 カメラ
31 未分化コロニー
32,33,34 分化コロニー
35,36 未分化コロニー
37 分化コロニー
44 ピペッティング装置
45 未分化コロニー
46 分化コロニー
47 剥離領域
48a,48b,48c 剥離領域
Claims (16)
- 多能性幹細胞からなるコロニーの撮像画像により、コロニーの識別を行う方法であって、前記撮像画像における輝度に基づいて、分化した多能性幹細胞を含む分化コロニーと未分化多能性幹細胞のみを含む未分化コロニーとを識別することを特徴とするコロニーの識別方法。
- 前記コロニーの輝度に基づいて、多層に重なった多能性幹細胞を含む多層コロニーを更に識別することを特徴とする請求項1に記載のコロニーの識別方法。
- 前記輝度の第1の閾値より明るい輝度の領域を有するコロニーを前記分化コロニーであると判断し、前記第1の閾値に等しいかこれより暗い輝度の領域のみを有するコロニーを前記未分化コロニーであると判断する請求項1に記載のコロニーの識別方法。
- 前記輝度の第1の閾値より明るい輝度の領域を有するコロニーを前記分化コロニーであると判断し、前記第1の閾値に等しいかこれより暗く第2の閾値に等しいかこれより明るい輝度の領域のみを有するコロニーを前記未分化コロニーであると判断し、前記第2の閾値より暗い輝度の領域を有するコロニーを前記多層コロニーであると判断する請求項2に記載のコロニーの識別方法。
- 前記第1の閾値は、前記撮像画像に一回又は複数回のメジアンフィルタによる処理を施すことにより得られるメジアンフィルタ画像について求めた各輝度値に対する画素数の分布に対して、判別分析法を適用することにより求められることを特徴とする請求項3に記載のコロニーの識別方法。
- 前記撮像画像に一回又は複数回のメジアンフィルタによる処理を施すことにより得られるメジアンフィルタ画像について求めた各輝度値に対する画素数の分布に対して判別分析法を適用することにより、前記第1の閾値が求められ、
前記撮像画像に一回又は複数回の平滑化フィルタによる処理を施すことにより得られる平滑化フィルタ画像の画素のうち、前記第1の閾値に105~115%の範囲の所定の倍率を乗じて得た閾値より小さい輝度値を有する画素のみ抽出することにより前記コロニー領域のみの画像を得、該コロニー領域のみの画像の各画素について輝度値を横軸とするとともに該輝度値を有する画素数を縦軸とするヒストグラムを求め、前記第1の閾値及び該ヒストグラムの最大値に相当する輝度値のうちの何れか小さい方の輝度値を最大輝度値とし、該最大輝度値より小さい輝度値の領域において、前記最大輝度値における画素数の90%の画素数から、前記最大輝度値の20%の画素数までの間に存在する前記ヒストグラム上の点の座標を用いて最小二乗法により直線を求め、該直線と前記横軸との交点を前記第2の閾値として求めることを特徴とする請求項4に記載のコロニーの識別方法。 - 請求項1乃至6の何れかに記載のコロニーの識別方法により前記未分化コロニーと該未分化コロニー以外のコロニーとを識別する工程、
前記未分化コロニー及び該未分化コロニー以外のコロニーの位置情報を取得する工程、
前記培養容器に細胞剥離剤を導入する工程、
前記位置情報に基づいて、前記未分化コロニーを剥離させる工程、及び
該未分化コロニーの剥離により得られる未分化多能性幹細胞を回収する工程
を包含することを特徴とする未分化多能性幹細胞の自動培養方法。 - 請求項1乃至6の何れかに記載のコロニーの識別方法により前記未分化コロニーと該未分化コロニー以外のコロニーとを識別する工程、
前記未分化コロニー及び該未分化コロニー以外のコロニーの位置情報を取得する工程、
前記培養容器に細胞剥離剤を導入する工程、
前記位置情報に基づいて、前記未分化コロニー以外のコロニーを剥離させる工程、
該未分化コロニー以外のコロニーの剥離により得られる多能性幹細胞を廃棄する工程、及び
前記未分化コロニーを剥離させて未分化多能性幹細胞を回収する工程
を包含することを特徴とする未分化多能性幹細胞の自動培養方法。 - 輝度値に基づいて画像処理された撮像画像を入力する画像入力手段と、
前記各コロニーの前記輝度に基づいて、分化した多能性幹細胞を含む分化コロニーと未分化の多能性幹細胞のみからなる未分化コロニーとを識別する識別手段と、
を備えたことを特徴とするコロニー識別装置。 - 前記識別手段は、前記コロニーの輝度に基づいて、多層に重なった多能性幹細胞を含む多層コロニーを更に識別することを特徴とする請求項9記載のコロニー識別装置。
- 前記識別手段は、前記輝度の第1の閾値より明るい輝度の領域を有するコロニーを前記分化コロニーであると判断し、前記第1の閾値に等しいかこれより暗い輝度の領域のみを有するコロニーを前記未分化コロニーであると判断することを特徴とする請求項9に記載のコロニー識別装置。
- 前記識別手段は、前記輝度の第1の閾値より明るい輝度の領域を有するコロニーを前記分化コロニーであると判断し、前記第1の閾値に等しいかこれより暗く第2の閾値に等しいかこれより明るい輝度の領域のみを有するコロニーを前記未分化コロニーであると判断し、前記第2の閾値より暗い輝度の領域を有するコロニーを前記多層コロニーであると判断することを特徴とする請求項11に記載のコロニー識別装置。
- 前記第1の閾値は、前記撮像画像に一回又は複数回のメジアンフィルタによる処理を施すことにより得られるメジアンフィルタ画像について求めた各輝度値に対する画素数の分布に判別分析法を適用することにより求められることを特徴とする請求項11に記載のコロニーの識別装置。
- 前記撮像画像に一回又は複数回のメジアンフィルタによる処理を施すことにより得られるメジアンフィルタ画像について求めた各輝度値に対する画素数の分布に判別分析法を適用することにより、前記第1の閾値が求められ、
前記撮像画像に一回又は複数回の平滑化フィルタによる処理を施すことにより得られる平滑化フィルタ画像の画素のうち、前記第1の閾値に105~115%の範囲の所定の倍率を乗じて得た閾値より小さい輝度値を有する画素のみ抽出することにより前記コロニー領域のみの画像を得、該コロニー領域のみの画像の各画素について輝度値を横軸とするとともに該輝度値を有する画素数を縦軸とするヒストグラムを求め、前記第1の閾値及び該ヒストグラムの最大値に相当する輝度値のうちの何れか小さい方の輝度値を最大輝度値とし、該最大輝度値より小さい輝度値の領域において、前記最大輝度値における画素数の90%の画素数から、前記最大輝度値の20%の画素数までの間に存在するヒストグラム上の点の座標を用いて最小二乗法により直線を求め、該直線と前記横軸との交点を前記第2の閾値として求めることを特徴とする請求項12に記載のコロニーの識別装置。 - 請求項9乃至14の何れかに記載のコロニー識別装置と、
前記培養容器に細胞剥離剤を導入する剥離剤導入手段と、
前記各コロニーの位置情報に基づいて、前記未分化コロニーを剥離させるとともに、該未分化コロニーの剥離により得られる未分化多能性幹細胞を回収するピペッティング装置と
を備えたことを特徴とする未分化多能性幹細胞の自動培養装置。 - 請求項9乃至14の何れかに記載のコロニー識別装置と、
前記未分化コロニー及び該未分化コロニー以外のコロニーの位置情報を取得する位置情報取得手段と、
前記培養容器に細胞剥離剤を導入する剥離剤導入手段と、
前記位置情報取得手段において取得した位置情報に基づいて、前記未分化コロニー以外のコロニーを剥離させ、該未分化コロニー以外のコロニーの剥離により得られる多能性幹細胞を廃棄し、更に前記未分化コロニーを剥離させて未分化多能性幹細胞を回収するピペッティング装置と、
を備えたことを特徴とする未分化多能性幹細胞の自動培養装置。
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JP5997826B2 (ja) | 2016-09-28 |
EP2487249A1 (en) | 2012-08-15 |
EP2487249B1 (en) | 2018-06-13 |
JPWO2011043077A1 (ja) | 2013-03-04 |
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