DE102018126486B4 - Viewing unit for cell analysis - Google Patents

Abstract

Observation unit for analyzing cells on a sample surface (25), with the following features:
a first housing part (110) having one or more windows (115);
a second housing part (120);
a sample holder (125) for receiving a sample (20) with the sample surface (25); and
an intermediate part (130) for providing a cavity (135) for cell analysis when the intermediate part (130) is arranged together with the sample holder between the first housing parts (110) and the second housing parts (120),
wherein the sample holder and the window (115) are arranged in such a way that an interaction between a cell culture introduced into the cavity (135) and the sample surface (25) can be observed via the window (115).

Description

The present invention relates to an observation unit for analyzing cells and to a method for observing cells on a sample surface, and in particular to a biological observation unit for analyzing cells on surfaces under physiological conditions.

BACKGROUND

When developing implants, the question of how the implant material or the implant surface interacts with the biological system, in particular human cells, regularly arises. For this purpose (in vitro) cell cultures of animal and human cells are used to gain initial insights into the effect of material properties on a real biological system (preferably human). For this purpose, cell cultures of human cells are often used in studies to obtain results on real human tissue. Here, it is examined how the materials or the surface of materials affect biological components (e.g. cell behavior, tissue, but also components produced by cells such as the extracellular matrix). These cell culture investigations are mostly designed for 2D investigations and do not allow a time-restricted viewing, which enables the analysis of phenomena such as cell migration or 3D interaction of different cell cultures, with non-transparent samples. They cannot depict the complex physiological state.

Although such cell culture studies provide initial results, they cannot reflect the behavior as tested in animal experiments due to the lack of complexity. Therefore, cell culture experiments are often only used for an initial test (e.g. for toxicity) of material properties before animal testing and before the clinical phase. However, the physiological difference between laboratory animals and humans is problematic for transferring the results obtained in animal experiments. In addition to fundamental ethical questions, animal experiments are also considerably more expensive than in vitro studies and do not allow for a finely timed study. Due to the large number of failed attempts (e.g. in animal experiments), the current development costs for new implants are very high.

The first few hours and days of ingrowth are the most important for analyzing how an implant works and should therefore be closely monitored in order to develop an understanding of the ingrowth processes. So far, this has not been possible using animal experiments, as there is no accessibility for observation.

There is therefore a need for viewing units and methods for observing cell cultures that overcome the above-mentioned problems and, in particular, allow the growth behavior of the cell cultures on sample surfaces to be monitored.

BRIEF DESCRIPTION OF THE INVENTION

At least part of the above problems are solved by a viewing unit according to claim 1 and a method according to claim 6. The dependent claims relate to advantageous developments of the subject matter of the independent claims.

Embodiments relate to a viewing unit for observing and analyzing cells or cell cultures on a sample surface. For this purpose, the cells can be embedded in a matrix that is located on the sample surface. The scope comprises: a first housing part with a window, a second housing part, a sample holder for receiving a sample with the sample surface, and an intermediate part for providing a cavity for cell analysis when the intermediate part together with the sample holder between the first housing parts and the second Housing parts is arranged. The sample surface and the window are arranged in such a way that an interaction between a cell culture introduced into the cavity and the sample surface can be observed via the window.

In particular, exemplary embodiments can use non-transparent samples, since the observation can take place from the opposite side (but can also take place from the side). The viewing unit is not limited to specific analyzes on cells or cell cultures, but allows all changes occurring in the cavity to be recorded. This includes not only analyzes of the cells, but also observations of their behavior (e.g. segregation of substances, changes in the environment caused by the cells, for example conversion of a matrix in which the cells are located). In addition, observation and analysis in the depth of the matrix is also made possible. Cell or matrix changes in areas that are not in direct contact with the sample surface are thus also recorded.

Optionally, the sample holder is part of the first housing part or the second housing part and is designed to interchangeably accommodate or hold different samples.

Optionally, the first housing part and/or the second housing part provides at least one connection channel in order to introduce a medium, in particular a nutrient solution, into the cavity from the outside and/or to remove another medium. For example, 2, 3, 4, ... connection channels can be provided for different purposes/examinations. A connection channel can be used for the supply of biological material (e.g. cells) or a biological component, another connection channel can be used for the supply of an analysis substance and another can be used as a discharge channel. The connection channels can all be formed on or in one housing part or be distributed over different housing parts.

Optionally, the cavity in the intermediate part has a height (in the direction perpendicular to the window) in a range from 100 µm to 700 µm (or between 150 µm to 400 µm or about 200 µm or even more, e.g. up to 3 mm or 10 mm) and is designed to enable two-dimensional (2-D) or three-dimensional (3-D) cell analysis.

Optionally, the viewing unit comprises at least one sealing element on at least one side of the intermediate part to seal the cavity.

Optionally, the viewing unit also includes at least one holder for at least one sensor or actuator, in order to enable further examinations of the cells or the cell culture. The further examinations can, for example, determine or change the pH value or generate a current or a magnetic field or carry out mechanical stimulation and record the reaction of the cells or cell matrix to it.

Embodiments also relate to a viewing system with at least two viewing units as previously described. The at least two consideration units can be arranged next to one another or connected to one another (e.g. in parallel or in series). The viewing system is therefore designed to record different samples or different cells or different cell cultures. The individual observation units can also have identical chambers with identical samples in order to examine different cell cultures in parallel under the same conditions. The viewing system can also be integrally integrated into an overall housing, with the first and second housing parts being able to be parts of the overall housing.

• - Bereitstellen einer Probe in einer Betrachtungseinheit, wie sie zuvor definiert wurde;
• - Einbringen einer Zellkultur in den Hohlraum der Betrachtungseinheit (z. B. in einem geeigneten Medium oder einer 3D-Matrix); und
• - Beobachten eines Zellwachstums oder einer Wechselwirkung zwischen der Zellkultur und der Probenoberfläche. (Wechselwirkung mit der Matrix, in der die Zellen eingebunden sind und die ebenfalls mit der Oberfläche interagiert);

Embodiments also relate to a method for analyzing and observing cells on a sample surface. The procedure includes the steps:

• - providing a sample in a viewing unit as previously defined;
• - introducing a cell culture into the cavity of the viewing unit (e.g. in a suitable medium or a 3D matrix); and
• - Observing cell growth or interaction between the cell culture and the sample surface. (Interaction with the matrix in which the cells are embedded and which also interacts with the surface);

• - zumindest teilweises Zuführen von Zellen über zumindest einen Verbindungskanal der Betrachtungseinheit;
• - Aufbringen der Zellkultur (z. B. Monokultur oder Kokultursysteme) auf die Probenoberfläche und Einbringen der Zellkultur zusammen mit der Probe in die Betrachtungseinheit;
• - Einbringen einer Suspension oder einer biologischen Probe oder 3-D Zellkulturmatrix (z. B. Kollagenmatrix), die die zu analysierenden Zellen aufweist.

Optionally, introducing the cell culture includes at least one of the following steps:

• - at least partially supplying cells via at least one connection channel to the viewing unit;
• - Applying the cell culture (e.g. monoculture or co-culture systems) to the sample surface and introducing the cell culture together with the sample into the observation unit;
• - Introduction of a suspension or a biological sample or 3-D cell culture matrix (e.g. collagen matrix) containing the cells to be analyzed.

• - Zeitliches Verfolgen des Zellwachstums oder des Anwachsens der Zellen an der Probenoberfläche (z. B. zeitliches Verfolgen des Zellverhaltens wie Formänderungen, Änderungen des Phänotyps, Wachstum, Migrationen, Zellmorphologie, Differenzierungen usw. in Gegenwart der Probe);
• - Zuführen von Nährstoffen, biologisch wirksamen Komponenten, Arzneimittel oder die Zellen beeinflussende Substanzen bzw. Komponenten über den zumindest einen Verbindungskanal;
• - Ermitteln einer Sterberate oder einer Teilungsrate oder einer Differenzierungsrate für die Zellen in der Zellkultur;
• - Wiederholen von Beobachtungen und Analysen für verschiedene Proben oder Probenoberflächen;
• - eine pH-Wertanalyse;
• - Untersuchung eines Zellverhaltens oder Zellmatrixverhaltens gegenüber Strom, Magnetfeld, mechanische Belastung etc.

Optionally, the monitoring includes at least one of the following steps:

• - Time tracking of cell growth or cell growth at the sample surface (e.g. time tracking of cell behavior such as shape changes, phenotype changes, growth, migrations, cell morphology, differentiations, etc. in the presence of the sample);
• - Supplying nutrients, biologically active components, drugs or substances or components influencing the cells via the at least one connecting channel;
• - determining a death rate or a division rate or a differentiation rate for the cells in the cell culture;
• - repeating observations and analyzes for different samples or sample surfaces;
• - a pH analysis;
• - Investigation of cell behavior or cell matrix behavior in relation to current, magnetic field, mechanical stress, etc.

Observing/analyzing should also refer to biological components. For example, cell behavior and cell growth as well as the interaction between the biological components (eg cells, metabolites, secreted components, extracellular matrix, etc.) and the sample, in particular the sample surface, are examined.

Exemplary embodiments solve at least some of the problems mentioned at the beginning by using a chamber (cavity) with a sample surface and the necessary connections, seals and the possibility of exact positioning, so that cell analyzes can be carried out under physiological conditions - comparable to animal experiments on a laboratory scale.

In particular, exemplary embodiments allow analyzes with regard to the influence of surfaces on biological systems such as cells, extracellular matrix, proteins, etc., since direct contact with a protein surface is possible under physiological conditions. By examining different surfaces and their influence on complex, time-dependent biological processes, relevant surface characteristics can be determined. Surface characteristics include, for example, topography, physical chemistry, stiffness and more.

A particular advantage of exemplary embodiments is that the observation unit enables the determination of data on the effect of surfaces that could otherwise only be determined in complex animal experiments or during clinical studies. Due to the direct usability of the results regarding the interaction between the cells and the surface, which can be determined continuously over a longer period of time, there is also the possibility of significantly expanding or optimizing the implant technology and the targeted functionalization. In addition, according to exemplary embodiments, it is possible to use the chamber for 2-D as well as for 3-D cell cultures and co-cultures.

Embodiments of the present invention achieve these advantages through the hollow space (chamber or cavity), which has an inflow (connecting channel) for a medium. The existing inflow can also be used to supply/supply the biological systems in the chamber with nutrients, active ingredients or analysis substances. In this way, physiological conditions can be created for the biological system to be considered.

BRIEF DESCRIPTION OF THE FIGURES

• 1 zeigt eine Betrachtungseinheit gemäß einem Ausführungsbeispiel der vorliegenden Erfindung.

The embodiments of the present invention will be better understood from the following detailed description and accompanying drawings of the various embodiments, which, however, should not be taken to limit the disclosure to the specific embodiments used for explanation and understanding only.

• 1 12 shows a viewing unit according to an embodiment of the present invention.

DETAILED DESCRIPTION

1 shows an embodiment of the observation unit for cell analyzes on a surface 25 of a sample 20. The observation unit comprises a first housing part 110, a second housing part 120, a sample holder 125, an intermediate part 130 and an optional sealing element 140. The sample 20 with the sample surface 25 is through the sample holder 125 is held between the first housing part 110 and the intermediate part 130, for example. The sample 20 itself does not need to be part of the observation unit, but can only be placed appropriately using the sample holder. For example, the sample holder 125 - as in the 1 shown - be an integral part of the second housing part 120 (but it does not have to be). It can also be part of the first housing part 110, in which case the second housing part 120 then only forms a cover as a closure or seal.

The first housing part 110 comprises one or more windows 115, which in the exemplary embodiment shown is designed as a transparent pane and can be inserted into an opening in the first housing part 110. The intermediate part 130 is placed on the transparent pane 115, the intermediate part 130 defining a cavity 135 through an elongated opening. An annular sealing element 140 is arranged on the intermediate part 130 and accommodates the sample 20 in the center or encloses it. The sample surface 25 is arranged on the side of the sample 20 facing the window 115, e.g. B. to be able to observe an increase in the cell culture on the sample surface 25 together with the growth of the cell culture.

Finally, the second housing part 120 is applied. The second housing part 120 can, for example, be firmly closed with the first housing part 110, for which purpose screw connections or the like can be provided, for example. One or more optional pins may prevent twisting of the first housing part 110 relative to the second housing part 120 . The second housing part 120 includes, for example, a holding structure for the sample 20 so that several or different samples can be placed one after the other within the second housing part 120 . In addition, the second housing parts 120 by way of example, a first and a second connection channel 121, 122, via which, for example, a medium can be introduced into or led out of the cavity 135 after the viewing unit has been assembled. There can also be more than two connecting channels. Likewise, at least some of the connection channels can be formed as part of the first housing part 110 . The medium can, for example, comprise a nutrient solution, pharmaceuticals, the cell culture itself or parts thereof, or other substances.

The sample 20 is made from an implant material, for example, and can have different surface structures that facilitate cell growth or the growth of cell cultures. The cell cultures can, for example, be applied to the sample surface before the observation unit is assembled. Optionally, it is also possible for the sample 20 to have several sample sections made of different materials or with different surface structures in order to carry out tests on different types of samples in parallel.

The cavity 135 in the intermediate part 130 is formed, for example, in such a way that the sample surface 25 forms a complete boundary above or on one side of the cavity 135 . The cavity 135 can extend laterally in the form of two elongate cavity extensions 136, 137 beyond the sample surface 25 in order to make contact with the first and/or the second connecting channel 121, 122. For this purpose, the sealing element 140 can, for example, have two through-openings 141, 142, through which the connecting channels 121, 122 are passed, in order to establish fluid contact with the cavity 135 after the viewing unit has been assembled.

The viewing unit shown is particularly suitable for observing and analyzing cell cultures and their interaction with the sample surface 25 during cell growth. The window 115 on the exemplary underside of the viewing unit allows continuous monitoring of the growth of the cell cultures. The at least one connection channel 121, 122 enables the supply/removal of a nutrient solution, medicinal product or other substances. In this way, for example, the pH value or saturation can be changed or inflammatory reactions can be triggered or correspondingly mapped/simulated. The diameter of the connecting channels 121, 122 is selected in particular so that no stresses in the medium supplied - especially if (further) cell cultures are to be supplied. Such stresses can otherwise have a negative impact on the resulting result.

1. a) wie oft teilen sich die Zellen (Ermittlung der Zellteilungsrate);
2. b) wie oft sterben die Zellen (Ermittlung der Sterberate);
3. c) wie gut differenzieren sich die Zellen (Ermittlung der Differenzierungsrate);
4. d) wie ist das Migrationsverhalten von Zellen.

A possible scheme for the cell analyzes to be able to assess the suitability of a surface can be carried out with regard to the following parameters, for example:

1. a) how often do the cells divide (determination of cell division rate);
2. b) how often the cells die (determination of the death rate);
3. c) how well the cells differentiate (determination of the differentiation rate);
4. d) how is the migration behavior of cells.

For example, before or during the examination, the cavity 135 can be (more or less) completely filled with a suitable cell culture matrix (e.g. collagens) or a suitable sample or suspension medium in which the cells are mixed or applied to a collagen surface . This makes it possible for both 2-D cell analyzes and 3-D growth to be examined. For this purpose, the cavity 135 can have a height in a range of 100 to 700 μm or approximately 200 μm (or up to 10 mm or up to 3 mm). The cell culture matrix can, for example, comprise a collagen 1A. However, another cell culture matrix can also be included, such as is produced during blood coagulation, for example.

However, the invention is not intended to be limited to a specific cell culture matrix (e.g., collagen or a collagen-free matrix) or to a specific application. Rather, the cell culture matrix is only used to cultivate the cells and should be suitable for this (i.e. include a suitable 3D cell culture matrix). The cavity 135 is intended to be restricted only to the extent that it allows direct contact of a sample surface 25 with the cells or extracellular components and also allows optical access through the window 115 for microscopic observation of the cell reactions at the surface 25 as a function of time.

• - Die Betrachtungseinheit erlaubt eine Zellanalysen, ohne dafür Tierversuche einzusetzen oder andere klinische Studien durchzuführen, da bereits im Vorfeld die Wirkung von Implantaten besser ermittelt werden kann. Um beispielsweise die Wirkungsweise von Implantaten mittels Tierversuche nachzuweisen, ist eine Bewertung zu mehreren Zeitpunkten unerlässlich, woraus sich eine erhebliche Anzahl von Versuchstieren ergibt.
• - Neben den Kosten für die Tierhaltung sowie der Durchführung der Versuche können Kosten für die histologische Aufbereitung der gewonnenen Präparate und deren Auswertung vermieden werden.
• - Da sowohl Tierversuche als auch klinische Studien bei der Neuentwicklung von Implantaten vorgeschrieben sind, besteht durch deren Substitution oder Verringerung oder durch eine gezielte Vorauswahl der nur vielversprechenden Implantate ein erhebliches Einsparpotenzial.
• - Im Gegensatz zu derzeit genutzten Betrachtungseinheiten, ermöglichen Ausführungsbeispiele die Betrachtung von Zellkulturen während die Zellkultur(en) mit einer Oberfläche 25 einer Probe 20 interagieren. Insbesondere kann ein Eindringen oder ein Anwachsen an Oberflächen unmittelbar beobachtet werden.
• - In der Kammer (Hohlraum 135) ist ausreichend Platz verfügbar, um Zellen gegenüber Medien oder Substanzen zu testen.

Exemplary embodiments offer the following advantages in particular:

• - The observation unit allows cell analysis without using animal experiments or conducting other clinical studies, since the effect of implants can be better determined in advance. For example, in order to demonstrate the mode of action of implants using animal experiments, an evaluation at several points in time is essential, resulting in a significant number of experimental animals.
• - In addition to the costs for keeping animals and carrying out the experiments, costs for the histological preparation of the specimens obtained and their evaluation can be avoided.
• - Since both animal experiments and clinical studies are required when developing new implants, there is considerable savings potential through their substitution or reduction or through a targeted pre-selection of only promising implants.
• - In contrast to currently used viewing units, exemplary embodiments allow for the viewing of cell cultures while the cell culture(s) interact with a surface 25 of a sample 20 . In particular, penetration or growth on surfaces can be observed directly.
• - Sufficient space is available in the chamber (cavity 135) to test cells against media or substances.

The features of the invention disclosed in the description, the claims and the figures can be essential for the implementation of the invention both individually and in any combination.

REFERENCE LIST

20

sample

25

sample surface

110

first housing part

115

Window

120

second housing part

121,122

one or more connecting channels

125

sample holder

130

intermediate part

135

cavity

136,137

Cavity extensions for the connecting channels

140

at least one sealing element

141, 142

Through openings for the connecting channels

Claims (10)

Observation unit for analyzing cells on a sample surface (25), with the following features: a first housing part (110) having one or more windows (115); a second housing part (120); a sample holder (125) for receiving a sample (20) with the sample surface (25); and an intermediate part (130) for providing a cavity (135) for cell analysis when the intermediate part (130) is arranged together with the sample holder between the first housing parts (110) and the second housing parts (120), wherein the sample holder and the window (115) are arranged in such a way that an interaction between a cell culture introduced into the cavity (135) and the sample surface (25) can be observed via the window (115). unit of consideration claim 1 , wherein the sample holder is part of the first housing part (110) or the second housing part (120) and is designed to accommodate or hold different samples interchangeably. unit of consideration claim 1 or claim 2 , wherein the first housing part (110) and/or the second housing part (120) provides at least one connection channel (121, 122) in order to introduce a medium, in particular a nutrient solution or components or substances to be tested, into the cavity (135) from the outside and/or to discharge another medium. A viewing unit according to any one of the preceding claims, wherein the cavity (135) in the intermediate part (130) has a height in a range from 100 µm to 10 mm and is configured to provide two-dimensional (2-D) or three-dimensional (3-D) enable cell analysis. A viewing unit according to any one of the preceding claims, further comprising at least one sealing element (140) on at least one side of the intermediate part (130) to seal the cavity (135). Observation unit according to one of the preceding claims, which further has at least one holder for at least one sensor and/or for at least one actuator in order to enable further examinations of the cells or the cell culture. Observation system with at least two observation units according to one of the preceding claims, wherein the observation units are connected to one another or are arranged next to one another and are designed to accommodate different samples or different cells or different cell cultures. A method for observing and analyzing cells on a sample surface (25), comprising the steps of: providing a sample (20) in a viewing unit according to any one of the preceding claims; introducing a cell culture into the cavity (135) of the viewing unit; and observing biological components in the cavity (135). procedure after claim 8 , wherein the introduction of the cell culture comprises at least one of the following steps: - at least partial supply of cells via at least one connection channel (121, 122) of the observation unit; - Applying the cell culture to the sample surface (25) and introducing the cell culture together with the sample (20) in the observation unit; - Introduction of a suspension or a biological sample or a 3-D cell culture matrix containing the cells to be analyzed. procedure after claim 8 or claim 9 , wherein the observation comprises at least one of the following steps: - observing and analyzing cell growth or an interaction between the cell culture and the sample surface (25); - observing an interaction of the cells with a surrounding matrix that interacts with the sample surface (25); - time tracking of the cell growth or the growth of the cells on the sample surface (25); - Supplying nutrients, biologically active components, drugs or substances influencing the cells via the at least one connection channel (121, 122); - determining a death rate or a division rate or a differentiation rate for the cells in the cell culture; - repeating observations and analyzes for different samples (20) or sample surfaces (25).

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