Micromagnetic resonance relaxometry (μMRR) for rapid and noninvasive detection of mesenchymal stem cell senescence

Abstract

Senescent cells can be detected using magnetic resonance relaxometry.

Description

[Technical Field]

[0001] (Priority Claim) This application claims priority to U.S. Provisional Application No. 63 / 272,738, filed October 28, 2021, which is incorporated herein by reference in its entirety.

[0002] (Technical field) The present invention provides systems and methods for non-invasive detection. [Background technology]

[0003] Cellular senescence is a cellular state in which aging and other external or internal conditions lead to cell cycle withdrawal, accompanied by genetic, metabolic, and morphological changes in cells (Reference 1). While senescence can be important for preventing cancer development and tumor progression (References 2-5), the accumulation of senescent cells in the body can have deleterious effects, particularly in age-related diseases such as neurodegeneration, cardiovascular disease, osteoarthritis, renal dysfunction, nonalcoholic fatty liver disease, and type 2 diabetes (References 3, 6, and 7). Cells with senescent characteristics are often present in tissues affected by age-related diseases, and their detection and elimination are major targets of medical intervention (Reference 2). Senescent cells exhibit reduced expression of genes involved in DNA replication, DNA repair, and the cell cycle (Reference 8). Furthermore, senescent cells exhibit senescence-associated secretory phenomena (SASP), which cause changes in the tissue microenvironment, local or systemic inflammation, and disruption of normal tissue architecture, leading to resistance of senescent cells to immune clearance (References 3, 6, and 9). Apart from aging, tumor-related stresses such as DNA damage (Ref. 10), reactive oxygen species (ROS) (Ref. 11), activation of certain oncogenes (Ref. 12), and exposure to interferon-β (Ref. 13) also induce cellular senescence (Ref. 2). Summary of the Invention [Means for solving the problem]

[0004] (overview) The presence of senescent cells in in vitro cultures of adult mesenchymal stromal cells (MSCs) has raised questions about their clinical utility in regenerative medicine and cell therapy. A method for noninvasive and rapid detection of senescent cells in heterogeneous cell cultures using microscale magnetic resonance relaxometry (μMRR) and a detection system has been reported. For example, T2 relaxation times measured with μMRR have been shown to strongly correlate with the proportion of senescent cells under diverse conditions, including different passages and donors, MSCs size-sorted by inertial spiral microfluidic devices, and senescence-induced MSCs. μMRR measurements also correlate well with traditional senescence detection assays, which often lead to cell destruction. Because the presence of senescent cells can affect the multilineage differentiation capacity and quality of MSCs, the μMRR assay and system described herein are promising, noninvasive, and rapid methods for critical quality analysis to improve MSC efficacy for patients.

[0005] Here, we describe a rapid and non-invasive method for detecting cellular senescence based on the transverse relaxation of protons using micromagnetic resonance relaxometry (μMRR).

[0006] In one embodiment, a method for detecting senescent cells can include loading a liquid sample containing a plurality of cells into a sensor, placing the sensor containing the liquid sample in a detection coil of a magnetic resonance relaxometry device, and determining a T2 value to detect the amount of senescent cells in the liquid sample. The sensor can be a tube or a chamber.

[0007] In another aspect, a method of improving the availability of stem or progenitor cells to a patient can include detecting senescent cells in a sample according to the methods described herein and enriching the cells in the sample to reduce the number of senescent cells in the sample.

[0008] In another aspect, a system for detecting senescent cells can include a magnetic resonance relaxometry device configured to detect the amount of senescent cells in a liquid sample and a cell separation device that reduces the amount of senescent cells in the liquid sample based on an output from the magnetic resonance relaxometry device. In certain circumstances, the liquid sample can be taken from a cell batch. The cell separation device can remove senescent cells from the cell batch based on detecting the amount of senescent cells.

[0009] In certain circumstances, the magnetic resonance relaxometry device can be configured to measure T2 values ​​to detect the amount of senescent cells in a liquid sample.

[0010] In certain circumstances, the amount of senescent cells in a liquid sample may be proportional to the ferritin concentration in the liquid sample.

[0011] In certain circumstances, the magnetic resonance relaxometry device can be configured to measure T2 values ​​to detect the amount of senescent cells, and Fe in the liquid sample. 3+ This includes quantifying the amount of

[0012] In certain circumstances, the amount of senescent cells in a liquid sample can be determined in comparison to a reference sample.

[0013] In certain circumstances, the amount of senescent cells in a liquid sample may be proportional to the ferritin concentration in the liquid sample.

[0014] In certain circumstances, determining the T2 value to detect the amount of senescent cells involves the use of Fe in the liquid sample. 3+ The method may include quantifying the amount of

[0015] In certain circumstances, the magnetic resonance relaxometry device may include a radio frequency probe.

[0016] In certain circumstances, the T1 relaxation time can be used in combination with the T2 value to determine whether senescent cells are present in a sample.

[0017] In certain circumstances, determining the T1 or T2 value may involve delivering a pulse train for a period of less than one minute.

[0018] In certain circumstances, determining the T1 or T2 value may involve acquiring and averaging between 2 and 70 scans.

[0019] In certain circumstances, T2 levels may decrease as the number of senescent cells increases.

[0020] In certain circumstances, the cells may include stem or progenitor cells. For example, the stem or progenitor cells may include mesenchymal stromal cells (MSCs), hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPSCs).

[0021] In certain circumstances, the detection volume of a magnetic resonance relaxometry device can contain a sample volume of less than about 1 μL, for example, less than 0.1 μL, less than about 0.01 μL, less than about 0.001 μL, or less than about 0.0001 μL, and in certain circumstances, the volume can be between about 1 pL and 10 pL.

[0022] In certain circumstances, determining T2 may include measuring the paramagnetic or ferromagnetic ion content of the cells. The paramagnetic or ferromagnetic ions may include iron or copper.

[0023] In certain circumstances, concentrating the cells may include passing the sample through a cell separator, thereby separating senescent cells from non-senescent cells in the sample.

[0024] In certain circumstances, the cell separation device may include a microfluidic cell sorter, an inertial focusing device, a microfluidic filtration device, a centrifugal flow device, a deterministic lateral displacement (DLD) chip, a tangential flow microfiltration device, or a combination thereof.

[0025] In certain circumstances, the cell separation device may include a microfluidic spiral path.

[0026] In certain circumstances, the liquid sample may be contained within a microcapillary.

[0027] Other aspects, embodiments, and features will become apparent from the following description, drawings, and claims. [Brief explanation of the drawings]

[0028] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] FIG. 1A shows a schematic diagram of a magnetic resonance relaxometry (MRR) device. [Figure 1B] FIG. 1B shows a schematic diagram of a system including a magnetic resonance relaxometry (MRR) device and a cell separation device. [Figure 2] Figure 2 shows the protocol for isolating senescent MSCs from an expansion culture and detecting their MRR. As shown in the figure, the spiral microfluidic device has one inlet and two outlets. MSC culture medium is pumped into the microfluidic device and sorted at a rate of 3.5 mL / min to collect large cells (22-26 µm) from the inner outlet. Cells are sorted again at a rate of 1.5 mL / min from the outer outlet to collect small, proliferating cells (11-15 µm) and medium-sized cells (15-22 µm). The normalized concentration of the sorted total cells is loaded into a microcapillary tube and analyzed by MRR. [Figure 3A] Figure 3A shows a schematic diagram illustrating the different behavior of iron ions in normal and senescent cells. [Figure 3B] FIG. 3B illustrates a pulse sequence that can be used with the systems and methods described herein. [Figure 3C] Figure 3C shows the MRR system and aging measurements. [Figure 4A] Figure 4A shows the separation of young / proliferating and senescent MSCs from an MSC expansion culture using a spiral microfluidic device. The MSC expansion culture is pumped into the microfluidic device and sorted at various speeds, and MSCs of different sizes are collected at the outlet. [Figure 4B] Figure 4B shows the cell diameter (μm) of MSCs sorted using the spiral microfluidic sorting device. [Figure 4C] Figure 4C shows the mean T2 values ​​(n = 6) of unsorted and size-sorted MSCs from two donors using the spiral microfluidic device. Large cells (22-26 µm) were size-sorted MSCs collected in the first round of sorting, while medium cells (15-22 µm) and small cells (11-15 µm) were collected in the second round of sorting. Statistical analysis of unsorted and small cells (n = 6, *P = 0.001) and unsorted and large cells (n = 6, *P = 0.002) was performed by two-tailed t-test. [Figure 4D] Figure 4D shows the mRNA expression levels of unsorted and size-sorted MSCs, demonstrating overexpression of senescence markers P16 and P21 in large MSCs. [Figure 4E] Figure 4E shows that Luminex assays of unsorted and size-sorted MSCs from two different donors demonstrate overexpression of the senescence-associated secretory phenotype. [Figure 4F] Figure 4F shows β-galactosidase staining of unsorted and size-sorted MSCs, with the cytoplasm of senescent MSCs stained blue (black arrows). [Figure 5A] Figure 5A shows the T2 values ​​(n=3) of MSCs from different donors D1 to D5 (passage 3). [Figure 5B] Figure 5B shows images of multilineage differentiation (adipogenesis, osteogenism, and chondrogenesis) of each MSC donor at passage 3. Osteogenic induction was confirmed by Alizarin Red S staining for calcium deposition, and adipogenic differentiation was confirmed by Oil Red O staining for lipid droplet detection. Chondrogenic differentiation was confirmed by glycosaminoglycan staining with Safranin O. [Figure 5C] Figure 5C shows the mean T2 values ​​(n=3) of MSCs serially passaged from 3 to 8. [Figure 5D]FIG. 5D shows the relative mRNA expression values ​​of P21 and P16 MSCs at passage P7 from the same donor compared with passage P4 as a control. [Figure 5E] FIG. 5E shows β-galactosidase staining of MSCs at passages P4, P6, and P8 from the same donor, showing blue staining of the cytoplasm of senescent MSCs (black arrows). [Figure 6A] Figure 6A shows immunofluorescence images of expanded MSCs from passages P3, P5, P6, and P3, as well as doxorubicin-treated MSCs. Cells are stained with a red-fluorescent senescence marker (γ-H2aX foci) and a green-fluorescent proliferation marker, 5-ethynyl-2'-deoxyuridine (Edu). Cell nuclei are counterstained with NucBlue (Hoechst 33342), which fluoresces blue (shown as DAPI) when bound to DNA. [Figure 6B] FIG. 6B shows β-galactosidase staining of MSCs at passages P3, P5, and P6, showing blue staining of the cytoplasm of senescent MSCs. [Figure 6C] [Figure 6D] Figures 6C and 6D show the proliferation (Pf) index (%) and senescence (Sn) index (%) calculated by counting the number of green and red fluorescent stains relative to the number of blue fluorescent stains in all images. A total of 10 images from each of three replicate staining experiments in P3, P5, P6, and DOX-treated MSCs were used to calculate the Pf index (%) and Sn index (%), which are plotted in Figure 6C and Figure 6D. [Figure 6E] Figure 6E shows the relative mRNA expression levels of senescence-associated markers p16 and p21 in MSCs at different passages: P3, P5, and P6. [Figure 6F] Figure 6F shows the average T2 results (n = 3) for P3, P5, P6, and DOX treatments. Statistical analysis of P3 and DOX (n = 3) was performed by two-tailed T-test in (Figure 6C), (Figure 6D), and (Figure 6F), and P values ​​are shown on each graph. [Figure 6G]Figure 6G shows the mean T2 values ​​(n = 3) of control MSCs (same donor, passage 4) treated with TGB-β1 (blue) and IL-1 (red) at different concentrations (10 and 20 mg / ml). [Figure 6H] Figure 6H shows the detection limit of the MRR assay for senescent MSCs. Shown are the mean T2 values ​​(n = 3) of MSCs at increasing cell numbers in microcapillary tubes containing control MRR and MSCs from the same donor treated with TGF-β1 (10 and 20 mg / ml) at passage 4. The T2 values ​​(n = 3) for PBS in microcapillary tubes are indicated by the black bars at the bottom of the figure (Figure 6B). [Figure 7A] Figure 7A shows the T2 values ​​of unsorted and sorted MSCs. Large cells (22–26 μm) are sorted MSCs collected in the first round of sorting, while medium cells (15–22 μm) and small cells (11–15 μm) are collected in the second round of sorting. [Figure 7B] Figure 7B shows images of multilineage differentiation (osteogenesis and chondrogenesis) of unsorted and sorted cells. [Figure 8] Figure 8 shows the detection limit of MRR for senescent MSCs. T2 values ​​are shown for various cell concentrations of control MSCs and MSCs (donor 3, passage 4) treated with TGF-β1 (10 and 20 mg / ml). The T2 value for PBS is indicated by the black bar at the bottom of the figure. DETAILED DESCRIPTION OF THE INVENTION

[0029] (Detailed explanation) Cellular senescence is a cellular state in which aging and other external or internal conditions lead to cell cycle termination, accompanied by genetic, metabolic, and morphological changes in the cell. (Reference 1) In addition to aging, tumor-related stresses such as DNA damage (Reference 10), reactive oxygen species (ROS) (Reference 11), activation of certain oncogenes (Reference 12), and exposure to interferon-β (Reference 13) can also induce cellular senescence. (Reference 2)

[0030] In addition to in vivo senescence observed in tissues, cellular senescence is also observed in cultured cells (Ref. 14). For example, mesenchymal stem / stromal cells (MSCs) have been intensively studied in numerous clinical trials for their potential application in ischemic, inflammatory, autoimmune, regenerative, and degenerative diseases (Ref. 9). However, senescent MSCs pose significant challenges for their clinical application (Refs. 14 and 15). Despite the clinical potential of MSCs, growth arrest, along with morphological and phenotypic changes, is observed in MSC populations after long-term in vitro culture (Refs. 14, 16-18). Furthermore, MSCs isolated from older donors exhibit higher levels of senescence than those from younger donors (Ref. 19). In senescent MSCs, cells become enlarged and flattened, with sclerotic nuclei and granular cytoplasm (Refs. 3, 14, 18, and 20). Aged MSCs have reduced multilineage differentiation capacity (References 14, 17, 18, and 21) and altered secretory and immunoregulatory functions, reducing their therapeutic value (References 3, 22, and 23).

[0031] The standard method for detecting senescent MSCs is histochemical staining for acid lysosomal β-galactosidase (β-gal), a cellular senescence biomarker (References 3 and 24). The staining procedure requires a stable pH (~6), cell fixation, and a long culture period (>12 hours). Alternatively, cellular senescence can be detected by estimating the remaining proliferative capacity of MSCs after multiple passages using a modified colony-forming unit (CFU) assay (References 18, 25, and 26). Real-time polymerase chain reaction (qPCR) can also detect senescence by providing gene expression levels of senescence-associated markers p16, p21, and p53 (References 3, 6, and 27). However, these assays require time-consuming and labor-intensive procedures (Reference 28). Additionally, these methods are destructive endpoint assays and are not suitable for quality control of MSCs during cell manufacturing. Therefore, a rapid detection method that can quantify the senescent state of MSCs in real time is important for quality control of regenerative medicine cell therapies.

[0032] There is significant evidence that iron homeostasis and its dysfunction are associated with aging and related conditions (Refs. 29-33). Cellular iron is expressed as Fe 2+ (reactive, unstable or "free" iron, diamagnetic) or Fe 3+ In iron-homeostatic cells, Fe is transported by various iron transport proteins (e.g., transferrin (Tf), TfR1, ferroportin, NCOA4, DMT1) and regulatory factors (e.g., p53, NRF2) (34). 2+ / Fe 3+ The uptake, balancing, and recycling of iron are maintained, and many different molecular pathways are involved (Ref. 35). Notably, increasing evidence indicates that increased intracellular iron is a universal feature of the aging phenotype and may serve as a "natural marker" of cellular senescence. Senescent cells regulate iron homeostasis proteins (Ref. 36) and also mobilize iron stores to Fe 2+ Ferritinophagy (reference 37) (Fe 3+ By inhibiting the autophagy of ferritin-bound Fe 3+ ) up to 30 times. 3+ Iron sequestration in labile iron (Fe ) released from ferritinophagy 2+ ) (Reference 39), and also prevents ferroptosis (Reference 38). 3+ ) may provide a noninvasive means for the detection of cellular senescence.

[0033] Microscale magnetic resonance relaxometry (μMRR) has previously been reported as an efficient method for diagnosing malaria (Ref. 40). Paramagnetic Fe in hemozoin crystals of infected red blood cells (RBCs) is detected. 3+ The increase in magnetic susceptibility of non-infected / healthy RBCs is due to the diamagnetic Fe 2+This resulted in a faster transverse relaxation (T2) of protons than the normal state (References 40 and 41). Furthermore, a μMRR-based phenotyping test for oxidative stress responses in the blood of diabetic patients was reported as an alternative to conventional A1c testing (Reference 42). Highly sensitive detection of tumor cells (References 43-45), bacteria (Reference 46), and Mycobacterium tuberculosis (Reference 47) using similar μMRR devices with immunomagnetically labeled molecular and cellular targets has also been reported. As demonstrated herein, μMRR is a non-invasive, label-free, and rapid method for detecting MSC senescence, enabling the identification of MSCs with limited proliferative capacity during MSC manufacturing for cell-derived products. This opens the door to broader application of this method to detect cellular senescence in other cells and tissues.

[0034] The systems and methods described herein are useful for detecting small numbers of cells (<10 5 ) and provides the ability to quantify senescent cells in a test tube by a rapid, non-invasive approach that requires no reagents or sample preparation steps. 3+ Direct quantification of paramagnetic Fe correlated unexpectedly well with traditional end-point, time-consuming assays commonly used to measure senescent cells, such as qPCR, Luminex assays, and β-galactosidase staining. Proton resonance relaxation measurements reveal the proportion of senescent cells in a sample. 3+ (diamagnetic Fe 2+ (except for ) increase the magnetic susceptibility and stimulate proton nuclear relaxation of intracellular water molecules, so this measurement is based on the Fe 3+ Previously, intracellular Fe 2+ (labile iron) has been measured by colorimetry and other assays (54), but Fe 2+ Due to the reactivity of Fe 2+ Reliable quantitative detection of Fe in a sample has generally been difficult (refs. 55 and 56). 3+ can be used to quantify the amount of Fe in a sample. 3+ The amount of may be correlated with cellular senescence.

[0035] An apparatus for performing magnetic resonance relaxometry is described, for example, in U.S. Patent No. 10,429,467, which is incorporated by reference in its entirety. As shown in FIG. 1A, the apparatus can include an MRR system. FIG. 1A is a schematic diagram of a magnetic resonance relaxometry (MRR) system 100 according to one embodiment of the present disclosure. The system 100 can include a field-programmable gate array (FPGA)-based (rf) spectrometer for controlling the MRR system 100, a first direct digital synthesis module for generating radio frequency pulses, a transmitter (TRANS) for transmitting the generated radio frequency pulses to a radio frequency (rf) probe and a detection coil 110, a receiver (RCVR) for receiving resonance information from the radio frequency probe, a first power amplifier (PA), a preamplifier (p-amp), a duplexer (Dup) for transmitting high-power excitation pulses to the radio frequency (rf) probe in a transmit mode and separating the high-power excitation pulses from the receiver in a receive mode, and a magnet system 120. The sample 130 can be placed in a sensor, such as a tube or chamber (e.g., a microcapillary tube), which can be placed in an RF detection coil. In many embodiments, the FPGA-based radio frequency (rf) spectrometer can include a pulse programmer (PPG) configured to control the FPGA-based radio frequency (rf) spectrometer and a second direct digital synthesis (DDS). The second DDS can generate a fixed intermediate frequency (IF). The first DDS can be configured to generate a variable desired frequency.According to one aspect of the present disclosure, the FPGA-based spectrometer can use the design described in "Takeda K. (2007), "A highly integrated FPGA-based nuclear magnetic resonance spectrometer," Rev Sci Instrum 78(3):033103" and / or "Takeda K. (2008) "OPENCORE NMR: open-source core modules for implementing an integrated FPGA-based NMR spectrometer," Journal of Magnetic Resonance 192(2):218-229," the teachings of these two references are incorporated by reference in their entirety.

[0036] To facilitate the processing of information to and from the MRR system 100, the FPGA-based radio frequency (rf) spectrometer is connectable to at least one external electronic device, including, for example, a personal computer, a mobile phone, and / or a portable electronic tablet. The connection between the MRR system 100 and the at least one external electronic device can be via at least one of USB, HDMI, and / or wireless connection means such as Wi-Fi and / or Bluetooth.

[0037] In conventional NMR systems, the primary cost of the equipment is the superconducting magnet (or permanent magnet) and radio frequency (rf) spectrometer. According to one aspect of the present disclosure, the overall system can cost less than $2500, with the majority of the cost coming from FPGA chips ($1000 each), external GHz clocks ($250 each), DDS (Analog Devices AD9858, $400 each), 1-watt power amplifiers ($100), preamplifiers ($50), receivers (RCVRs) (AD8343, $4 each), transmitters (TRANS) (AD834, $20 each, and AD8343), and USBs (FT2232D, $10 each). The costs of the major electronic components used are listed in parentheses. Other peripheral components, such as pin connectors (e.g., SMA), capacitors, rf switches, rf transformers, and rf filters, cost less than $10 each.

[0038] The MRR system 100 can be adapted to operate in various modes to detect NMR-active nuclei, such as protons, fluorine, phosphorus, and carbon. The magnetic field used in each mode in which the MRR system 100 operates varies depending on which nuclei are being detected. Depending on the mode of operation, the MRR system 100 can operate in a magnetic field of approximately 0.1 to 3 Tesla (T), which corresponds to a frequency of approximately 1 to 150 MHz. For example, when the MRR system 100 is operating in proton NMR mode, the magnetic field is approximately 0.76 T, which corresponds to a proton NMR frequency of approximately 31.9 MHz.

[0039] The MRR system 100 can be controlled by an FPGA-based radio frequency (rf) spectrometer consisting of a pulse programmer and a second DDS. Compared to CMOS technology, FPGAs offer the advantage of being reprogrammable. FPGA-based radio frequency (rf) spectrometers can be programmed using tools and software provided by vendors such as Altera Corporation, San Jose, California, USA, and Xilinx, Inc., San Jose, California, USA. In an exemplary embodiment, the FPGA chip can include an EP3C80F780C8N (Altera Cyclone III) embedded in a breadboard (ACM-202-80C8, Human Data Corporation, Japan). This chip has 81,000 logic elements and, when fully utilized, can generate three independent if outputs.

[0040] The pulse programmer generates high-power excitation RF pulses. The RF pulses then pass through a first power amplifier, which generates optimized RF power for approximately 1-1000 microseconds to effectively excite all nuclei. The high-power RF pulses are then transmitted to an RF probe, as further described herein.

[0041] In exemplary operation, the power used for liquid-state and solid-state NMR is approximately 0.1 W to 10 W and approximately 100 W to 1000 W, respectively. While a "powerful" power amplifier is often essential for MRR systems, such amplifiers are bulky and require high power consumption, severely limiting field operation. For example, a novel and lightweight 1-watt power amplifier can be constructed with a 4 cm x 4 cm printed circuit board. A solenoidal microcoil (inner diameter 700 to 1000 μm, e.g., 750, 800, 850, 900, or 950 μm) can be further employed to generate a strong oscillating magnetic field B1 and pick up signals from free induction decay (FID) or spin echoes. By employing a duplexer, the high-power excitation IF pulses transmitted to the RF probe in transmit mode can be separated from the receiver or detection coil 110 during receive mode. The FID / spin echo is then amplified by a preamplifier (AMP-75+, Mini Circuits, USA) with a gain of 20 dB and a noise figure of 2.83, and finally filtered by an appropriate low-pass filter before being sent to the receiver circuit. The FID is the observable NMR signal generated by non-equilibrium nuclear spin magnetization rotating around the static magnetic field (conventionally the z-axis). This non-equilibrium magnetization can be induced by applying a resonant radio frequency pulse close to the Larmor frequency of the nuclear spins. The spin echo is a refocusing pulse consisting of one 90° reversal followed by a 180° reversal at resonance.

[0042] The magnet system 120 is portable and lightweight (e.g., approximately 60 g) and can be configured to generate a high static magnetic field. The magnet system 120 can include at least one magnet positioned adjacent to the RF probe. Alternative embodiments include having at least two magnets positioned adjacent to the RF probe. The RF probe can be positioned between the at least two magnets. The magnet system 120 can include permanent magnets and / or electromagnets. Permanent magnets used in the magnet system 120 can include, for example, neodymium-based magnets.

[0043] The detection region of the magnetic resonance relaxometry device can contain a sample volume of less than about 1 μL for detection. For example, the sample can be provided within a sensor, such as a capillary tube, a microcapillary tube, or a chamber. Sealing the sample from the ambient environment can reduce exposure to oxygen and other substances that may adversely affect the ability of the magnetic resonance relaxometry device to detect senescent cells.

[0044] As described herein and based on the device description above, a method for detecting senescent cells can include loading a liquid sample containing a plurality of cells into a sensor, placing the sensor containing the liquid sample in a detection coil of a magnetic resonance relaxometry device, and determining a T2 value to detect the amount of senescent cells in the liquid sample. Details of determining the T2 value to detect the amount of senescent cells are described below. For example, the T2 value may decrease as the number of senescent cells increases. In certain circumstances, determining the T2 value can include measuring the magnetic susceptibility index of the cells.

[0045] Current μMRR setups are only suitable for measuring cell suspensions in liquid samples. However, μMRR setups can be designed for adherent cells on cell culture plates. The device, system, and method described herein facilitate the removal of senescent cells from cell populations by simplifying the identification process. This approach, combined with cell manipulation techniques, can be used to develop and improve cell therapy.

[0046] In certain instances, the amount of senescent cells in a liquid sample can be determined by comparing it to a reference sample. The device can be calibrated to quickly and reliably measure the amount of senescent cells in a liquid sample. Alternatively, the device can compare two samples to determine the relative amount of senescent cells in the liquid sample. For example, two samples can be distinguished between those with a high and a low relative concentration of senescent cells.

[0047] In certain instances, the amount of senescent cells in a liquid sample can be proportional to the concentration of ferritin in the liquid sample. As described below, the unexpected relationship between the amount of ferritin in a liquid sample and the amount of senescent cells in the liquid sample is one feature that facilitates using a magnetic resonance relaxometry device to monitor, measure, or determine the presence of senescent cells in a sample.

[0048] One method for determining T2 values ​​to detect the amount of senescent cells involves measuring Fe in a liquid sample. 3+ In certain circumstances, determining T2 may include quantifying the amount of other paramagnetic or ferromagnetic ions in the cells (e.g., copper, specifically copper ions). Other paramagnetic or ferromagnetic ions may include cobalt, nickel, or manganese, cobalt ions, nickel ions, or manganese ions. The magnetic resonance relaxometry device may measure Fe 3+ For example, determining the T1 or T2 value can include delivering a pulse train over a period of less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute.

[0049] Various approaches can be taken to improve the accuracy of detecting senescent cells. For example, determining the T1 or T2 value can include acquiring and averaging multiple scans. Up to 100 (or more) scans can be averaged. The number of scans can be less than 80, less than 70, or less than 60. More typically, 10 to 50 scans can be averaged. At least 2, 4, 5, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 scans can be averaged under certain circumstances. In certain circumstances, the T1 relaxation time can be used in combination with the T2 value to determine whether senescent cells are present in a sample.

[0050] The devices, systems, and methods described herein can be used to monitor or analyze the senescence of many different cell types. Identifying senescent cells in a cell population can allow the population to be purified or purified to remove the senescent cells, thereby improving the productivity of the cell population for a desired application, such as therapy. For example, the cells can include stem or progenitor cells. For example, the stem or progenitor cells can include mesenchymal stromal cells (MSCs), hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPSCs). For example, mesenchymal stromal cells (MSCs) can be used more effectively in a desired cell therapy.

[0051] The above-described system can be used to improve the efficacy of mesenchymal stromal cells in a patient. The method for improving efficacy can include detecting senescent cells in a sample as described above and enriching the cells in the sample to reduce the number of senescent cells in the sample. The cell separation device can be a membrane-containing cell separation system, a sequestration agent for removing senescent cells, or a microfluidic device. For example, enriching the cells can include passing the sample through the cell separation device.

[0052] A cell separation device, such as a microfluidic spiral path device, can be used to separate senescent cells from non-senescent cells in a sample. The cell separation device can include a microfluidic cell sorter, an inertial focusing device, a microfluidic filtration device, a centrifugal flow device, a deterministic lateral displacement (DLD) chip, a tangential flow microfiltration device, or a combination thereof. The cell separation device can remove senescent cells based on monitoring the senescent cells. For example, the cell separation device can remove senescent cells from a cell batch (e.g., a cell batch from which a liquid sample was taken) based on detecting the amount of senescent cells.

[0053] 1B, the systems described herein can include a magnetic resonance relaxometry device 200 and a cell separation device 240. For example, a system for detecting senescent cells can include a magnetic resonance relaxometry device configured to detect the amount of senescent cells in a liquid sample and a cell separation device that reduces the amount of senescent cells in the liquid sample based on an output from the magnetic resonance relaxometry device.

[0054] We describe the non-invasive and rapid detection of senescent MSCs using magnetic resonance relaxometry. As shown in Figure 3A, we unexpectedly discovered that the chemical nature of iron ions differs between normal and senescent cells. Normal cells maintain iron homeostasis mediated by numerous iron transporters and iron-binding proteins. Senescent cells exhibit paramagnetic Fe 3+ This correlates with the accumulation of iron (inactive form). For example, normal cells have Fe 2+ and Fe 3+ However, senescent cells have a high level of Fe 3+ Accumulate.

[0055] As shown in Figure 3B, the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence for measuring T2 values ​​can function efficiently in the inhomogeneous magnetic field generated by the permanent magnets of the MRR. A train of radiofrequency pulses is irradiated onto proton nuclei with a resonance frequency of 21.65 MHz, with an inter-echo time interval of t echoes. This sequence repeats thousands of echoes until relaxation occurs over time. The height of this decaying echo is called the transverse relaxation time (T2).

[0056] As shown in Figure 3C, the MRR system can be configured with a portable permanent magnet that provides a strong magnetic field. A homemade radio frequency (RF) detection probe is connected to an RF spectrometer. For T2 measurements, MSC samples (young / proliferating / senescent MSCs) in microcapillary tubes can be placed in the RF detection coil. The microcapillary tube contains 4 μl of MSC sample within a 4 mm detection range of the RF detection coil. The typical required cell number is 60,000 MSCs within the detection volume. The bright areas are crysseals that seal the microcapillary tube. The right side shows the results of the measurements of young and senescent MSCs. 1 H spin-spin relaxation time T2 is shown, and T2 is decreased in aged MSCs compared to young MSCs.

[0057] result MRR assay confirmed high levels of senescence in large MSCs enriched by size sorting using a spiral microfluidic device

[0058] In preclinical and clinical studies, the formation of senescent cells in MSC cultures often reduces the therapeutic efficacy of the cells. Previously, cell size, along with other biophysical characteristics, was identified as a non-invasive marker for distinguishing MSC phenotypes (References 48 and 49). Small MSCs are generally multipotent, whereas large MSCs tend to be proliferation-restricted and differentiate more significantly toward the osteogenic lineage. Large MSCs sorted by inertial microfluidic devices were found to exhibit a senescent phenotype with limited proliferative capacity and a longer doubling time compared to small MSCs (Reference 50) (Figures 4A–4F). MRR assays, based on size sorting using a spiral microfluidic device, indicate that large MSCs are more senescent. Using the same microfluidic device, MSCs were sorted into three size groups: unsorted, small (11–15 μm), medium (15–22 μm), and large (22–26 μm) (see Figure 4A and 4B). The size-sorted cells were then analyzed using a μMRR (Figure 4C). Namely, MSCs were added to a microcapillary tube and placed in the radio frequency (RF) detection coil of the μMRR device for T2 measurements. MRR analysis of sorted MSCs showed a decrease in T2 values ​​from small to large cells (Figure 4C), indicating that the proton nuclear relaxation of water, which is ~2000 ms in pure water, is related to the paramagnetic iron (Fe) in senescent MSCs. 3+ ) impurities significantly reduced the expression of senescence-related markers p16 and p21 (measured by qPCR) in large cells compared to the medium and small groups (Figure 4D). Furthermore, β-gal staining (Figure 4E) strongly correlated with both MRR and qPCR results. Consistent with the lower T2 values ​​measured by μMRR, higher intensity β-gal staining was observed in large group cells and unsorted group cells. These results suggest that Fe in senescent cells 3+ This supports previous reports on the preservation of T2 relaxation (ref. 36), because the faster T2 relaxation is due to the presence of paramagnetic Fe 3+This is due to increased secretory content. Secretion of molecules associated with the senescence-associated secretory phenotype (SASP) was also measured in unsorted and size-sorted MSC subpopulations (small, medium, and large) (Figure 4D). High expression of key secretory markers associated with SASP was observed in larger MSCs. See, for example, Lu Yin, Zheng Yang, et al., Biomaterials, 240, 119881 (2020), incorporated by reference in its entirety.

[0059] MRR-based senescence measurements correlate with changes in the multilineage differentiation capacity of MSCs from different donors

[0060] MRR analysis measures changes in multilineage differentiation in MSCs from different passages and donors. A key therapeutic quality of MSCs is their ability to undergo multilineage differentiation into cell lineages such as adipocytes, chondrocytes, and osteoblasts. MSCs from different donors vary in their multilineage differentiation potential, which has been an important but poorly understood limitation in achieving consistent quality control of therapeutic MSCs. Here, we analyzed MSCs cultured in vitro up to passage 3 from five different donors (D1–D5; see Methods for donor description). For MRR analysis, 6 × 10 cells were used for each T2 experiment. 4 Normalized cell counts were used. Figure 5A shows the T2 profiles of MSCs from different donors. The multilineage differentiation potential of MSCs from corresponding donors (D1 to D5) was measured by staining (Figure 5B). Analysis of differentiation potential revealed that D1 MSCs possessed full multilineage differentiation potential, while D5 MSCs possessed the most limited potential. The T2 values ​​of MSCs from each donor correlated with their overall differentiation potential, with the highest T2 values ​​observed for MSCs from D1, whereas the lowest T2 values ​​were measured for MSCs from D5. Thus, lower T2 values ​​corresponded to reduced multilineage differentiation potential. The reduced multilineage differentiation potential of MSCs from certain donors may be influenced by the high proportion of senescent cells in the population, which may also affect cell proliferation.

[0061] MRR analysis shows that serial passaging of MSCs increases cellular senescence

[0062] It is possible to correlate the MRR with standard assays. During in vitro culture of MSCs, cellular senescence tends to increase during serial passaging. Senescent cells can affect MSC proliferation and differentiation capacity, and ultimately the quality of cells for clinical use. Therefore, it is important to rapidly identify and potentially remove senescent cells from each passage of MSC culture (Reference 50). Next, MSCs from donor 1 were analyzed under in vitro culture conditions from serial passages P3 to P8. Using the μMRR, the normalized T2 values ​​of MSCs from each passage are shown in Figure 6G. The highest T2 values ​​of MSCs were observed in early passages, but these levels gradually decreased with later passages. Based on the intensity and number of cells showing β-gal staining, MSCs from later passages, which contained a greater proportion of senescent cells, exhibited the lowest T2 values ​​(Figure 6H). Similarly, qPCR of the cell cycle inhibition markers p16 and p21 showed the highest expression in cells from later passages compared to early passages (Figure 6E).

[0063] Senescence detection by immunofluorescence assay and MRR

[0064] To directly correlate cell status with the MRR T2 value, proliferation and senescence were simultaneously measured. MSCs from the same donor were serially passaged to passages 3, 5, and 6. As a positive control for senescent cells, passage 3 cells were also treated with doxorubicin (DOX). DOX is a chemotherapeutic agent that generates DNA damage and reactive oxygen species, inducing senescence in tumor cells. To measure DNA synthesis, EdU (5-ethynyl-2'-deoxyuridine) was used as an indicator of active cell proliferation (shown in green). In contrast, senescent cells are known to accumulate γ-H2AX foci (shown in red). The proliferation (Pf) and senescence (Sn) indices were calculated from these images and plotted, respectively (Figure 6C and Figure 6D). MRR experiments were also performed on cells from all passages and DOX-treated cells, and the T2 values ​​for each condition are shown (Figure 6F). The staining images and Pf / Sn index graphs revealed that there were more proliferating cells (EDU) in P3. Pf gradually decreased in later passages (P5 and P6) and was lowest in DOX-treated cells. The Sn index showed the opposite trend, with more senescent cells (γ-H2aX) observed in P6 and DOX-treated cells compared to P3 and P5. These results were strongly consistent with the T2 measurements (Figure 6F), confirming that the MRR assay is a robust method for noninvasively detecting senescent cells. In addition, the proportion of β-gal-stained cells was higher in later passages (P6), consistent with the T2 results from each passage. Similarly, high expression of p16 and p21 was observed in MSCs from P6 by qPCR (Figure 6E), confirming the μMRR measurements.

[0065] Senescence induction by cytokine treatment

[0066] Cytokines such as transforming growth factor-β1 (TGFβ1) and interleukin-1 (IL-1) induce senescence in MSCs. TGFβ1 is a member of the TGF family that regulates many cellular functions, including proliferation and cell death (References 51 and 52). IL-1 is a proinflammatory cytokine whose expression is associated with SASP (Reference 53). Therefore, we treated MSCs with different concentrations of TGFβ1 and IL-1 (10 and 20 ng / ml) and measured their T2 levels using the mMRR approach. The T2 levels of TGFβ1- and IL-1-treated cells decreased with increasing cytokine concentrations. Senescent cells were more prevalent in TGF-β1-treated cells than in IL-1-treated cells (Figure 8), which was confirmed by the decreased T2 levels compared with IL-1-treated cells.

[0067] Because MSC donors vary in their proliferation capacity, it is important to determine the limit of detection (LOD) of the MRR assay in terms of cell number. For all μMRR experiments, the normalized cell number was 6 × 10 in a 4 μl volume in a microcapillary tube. 4 Here, as shown in Figures 6H and 8, the cell concentration was further reduced to 4 × 10 for μMRR detection. 4 , 2 × 10 4 , 1×10 4 The LOD was analyzed by reducing the number of cells. MSCs were treated with different concentrations of TGFβ1 (10 and 20 mg / ml) to induce senescence, and these cells were harvested for MRR detection. TGF-β1-treated and untreated MSCs were analyzed by reducing the number of cells to 1, 2, and 4 (×10) in a detection volume of 4 μl. 4 A batch of 1 x 10 cells was analyzed by MRR. The T2 values ​​of PBS were also compared to the T2 values ​​of MSCs (treated and untreated). In this analysis, the T2 values ​​of 1 x 10 cells were compared to the T2 values ​​of untreated MSCs and PBS. 4 Even at the lowest cell concentrations, we demonstrate significant differences in T2 values ​​of senescent MSCs. Collectively, the data described herein suggest that MRR can be used to noninvasively measure the presence of senescent cells in heterogeneous cultures, which may be employed to improve bulk culture of clinical-grade cell therapy products such as MSCs.

[0068] A new method for quantifying senescent cells in vitro has been established. This method is rapid, non-invasive, and requires a small number of cells (<10 5 ) and does not require any reagent or sample preparation steps. 3+ Direct quantification of paramagnetic Fe correlated unexpectedly well with traditional end-point, time-consuming assays commonly used to measure senescent cells, such as qPCR, Luminex assays, and β-galactosidase staining. 3+ (diamagnetic Fe 2+ (except for ) increase the magnetic susceptibility and stimulate proton nuclear relaxation of intracellular water molecules, so this measurement is based on the Fe 3+ Previously, intracellular Fe 2+ (labile iron) has been measured by colorimetry and other assays (54), but Fe 2+ Due to the reactivity of Fe 2+ Reliable quantitative detection of Fe has generally been difficult (refs. 55 and 56). 3+ Ferritin is often used as a surrogate marker for the quantification of Fe (Reference 57). Ferritin contains up to 4500 atoms of Fe. 3+ Ferritin is a large (~480 kDa) protein complex that can accommodate iron, but its concentration varies depending on the amount of Fe stored in the cell. 3+ ICP-MS quantification of total iron concentration (58) is the current standard for iron quantification, but this does not correlate well with accumulated Fe. 3+ may not be accurately reflected.

[0069] In contrast to traditional iron quantification methods, MRR is uniquely positioned to advance general iron biology. μMRR allows for complete, noninvasive phenotyping of senescent cells and allows for downstream biological and functional measurements of other indicators on the same cells. This capability allows for robust and direct correlations to be made between MRR and other biochemical and functional measurements. This approach is potentially transformative for the field, especially given the lack of consensus on cell surface markers specific to senescent cells.

[0070] Additionally, the "iron imaging" modality utilized here is already widely used in T1- and T2-weighted MRI imaging (Refs. 59-62). Therefore, findings from MRR quantification of cellular senescence can be easily compared with in vivo pathology. Given that the resolution of modern MRI approaches is less than 100 μm (Ref. 63), noninvasive detection of senescent cells in living tissues may be feasible in the future. Most importantly, the ability to quantify the degree of MSC senescence will unparalleledly enable the therapeutic application of MSCs. Variability in MSC quality, primarily due to differences in the degree of senescence, presents a significant unresolved bottleneck. μMRR is envisioned as a rapid, noninvasive quality screening tool for MSCs in culture-based production processes.

[0071] In this study, the multilineage differentiation potential of MSCs from different donors also correlated well with the MRR results, with the highest T2 values ​​measured from MSCs with strong multilineage differentiation potential. This technology will be useful for real-time screening of MSCs with the highest proliferation and differentiation potential in a patient-specific manner. This new bioengineering modality can also be applied to optimize the biomanufacturing of MSCs or other cell therapy products. For example, MRR can be used to monitor cell cultures in real time in a way that allows senescent cells to be removed using microfluidic sorting (Ref. 50). Therefore, this approach has the potential to overcome a major bottleneck in the field to produce higher-grade MSCs and cell products for therapeutic applications.

[0072] Methods and Materials MSC culture

[0073] Bone marrow-derived mesenchymal stem cells (MSCs) were purchased from Lonza Pte Ltd and Rooster Bio Inc. Cells were cultured in low-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% glutamax, and 1% penicillin / streptomycin (Thermo Fisher Scientific, Singapore) at an initial cell density of 1500 cells / cm at 37°C in a CO2 5% atmosphere. 2MSCs were grown in tissue culture plates (TCP). The medium was changed every two days, and cells were harvested at 80% confluency for further experiments or subculture. During cell harvesting, cells were incubated with 0.25% trypsin-EDTA (Thermo Fisher Scientific, Singapore) for 3 minutes. Cell counts were calculated by trypan blue exclusion using a disposable hemocytometer (INCYTO, Korea). Unless otherwise specified, MSCs from passage 3 (P3) to P5 were used for further experiments. MSC senescence induction was performed by treating them with medium containing 5, 10, or 20 ng / ml TGF-β1 for 3 days. For doxorubicin (DOX) treatment, cells were grown at a density of 1500 cells / cm. 2 and incubated with 1 μM doxorubicin (Sigma) at 37°C for 24 hours. The medium was then removed and fresh medium was added to the cells, which were then incubated for 24 hours before analysis.

[0074] MSC sorting by inertial spiral microchannel device

[0075] The inertial spiral microchannel device was designed and fabricated using the same method as previously described (50). The device has eight loops with radii decreasing from 12 mm to 4 mm and a trapezoidal cross section with a width of 580 μm, an inner diameter of 85 μm, and an outer height of 133 μm. It has one inlet for the introduction of the cell suspension to be sorted and two outlets for the collection of sorted cells. The design was engraved into a micromachined aluminum mold (Whits Technologies Inc., Singapore), and the device was cast from a 10:1 mixture of base and curing agent (Sylgard 184, Dow Corning Inc., USA) in polydimethylsiloxane (PDMS).

[0076] Prior to sorting, MSCs were resuspended in culture medium at 1–2 million cells / ml and loaded into a syringe (Thermo Fisher Scientific, Singapore) connected to Tygon tubing (Spectra Teknik Pte Ltd., Singapore). One tube was inserted into the inlet of the device, and two separate tubes were inserted into the outlets to collect the sorted cells. A syringe pump (PHD2000, Harvard Apparatus Inc., USA) was used to control the flow rate of the cell suspension within the device. Two sequential sorting steps were performed to separate MSCs into three size-dependent subpopulations. The first sorting was performed at 3.5 ml / min, with the largest subpopulation (22–26 μm) collected at the inner outlet. The cells collected at the inner outlet were subjected to a second sorting step at 1.5 mL / min. The cells collected at the inner outlet and the outer outlet were sorted into medium-sized subpopulations (15-22 μm) and smallest subpopulations (11-15 μm), respectively. Unsorted MSCs were used as a control in this experiment. Separation within the inertial spiral microchannel device was visualized in real time using an inverted microscope (IX71, Olympus Co., Japan) equipped with a high-speed CCD camera (Phantom v9, Vision Research Inc., USA).

[0077] MRR measurement

[0078] The MRR consists of a portable permanent magnet with B = 0.5 T (Metrolab Instruments, Plan-les-Ouattes, Switzerland) and a tabletop NMR console (Kea Magritek, Wellington, New Zealand). 1H MRR measurements were performed at a resonant frequency of 21.65 MHz inside the magnet. A single-resonant proton MRR probe with a 900-μm inner diameter detection microcoil was used to house the MRR sample in a microcapillary tube (outer diameter: 1,500 μm, inner diameter: 950 μm) (22-260-950, Fisherbrand, Waltham, MA, USA). The MRR probe's electronics and coil were mounted on a single printed circuit board (Figure 3C). (Ref. 40) All experiments were performed at 26.3 °C inside the magnet, maintained by a temperature controller (RS Components, UK).

[0079] In all MRR experiments, unless otherwise stated, a normalized concentration of MSCs (3 × 10 5 MSC samples were spun down at 300 g for 5 min, and the supernatant was aspirated. The pellet was suspended in 20 μl of PBS and loaded into a 4-mm-long microcapillary tube. The microcapillary tube was sealed with a crysseal and attached to a coil for MRR measurements. The proton transverse relaxation rate (T2) was measured using a standard CPMG (Carr-Purcell-Meiboom-Gill) pulse program (references 64 and 65) (Figure 3B). The transmitter power output was maintained at 12.5 mW with a single 90° pulse of 16 μs pulse length for all T2 measurements. A CPMG pulse train with an echo count of 4000 and an inter-echo time of 60 μs was used in all experiments. A 2-second recycle delay was used, sufficient time for all spins to return to thermal equilibrium. For signal averaging, 24 scans were performed in all experiments.

[0080] Real-time polymerase chain reaction (qPCR) analysis

[0081] Total RNA was extracted with the RNeasy Mini kit (Qiagen, USA) according to the manufacturer's protocol. The RNA concentration was measured using a NanoDrop UV-vis spectrophotometer (NanoDrop Technologies, USA). Then, the RNA was analyzed using iScript TM200 ng of total RNA was reverse transcribed using a cDNA synthesis kit (Biorad Laboratories, USA). Real-time PCR was performed using the SYBR Green system with the primers shown in Table 1. Real-time PCR was performed at 95°C for 10 minutes, followed by 40 cycles of amplification, including a denaturation step at 95°C for 15 seconds and an extension step at 60°C for 1 minute, using an ABI 7500 Real-Time PCR System (Applied Biosystems, USA). Gene expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the respective control groups, using the formula: 2 -ΔΔCt was calculated using

[0082] Table 1. Real-time PCR primers

[0083] [Table 1]

[0084] Conditioned medium and ELISA

[0085] Size-sorted MSCs were seeded into T75 flasks and allowed to adhere overnight before removing the medium the next day. Cells were washed once with PBS and cultured in serum-free medium for 48 hours at 37°C and 5% CO2. The medium was then collected and centrifuged at 4500 x g for 15 minutes to remove cellular debris. The supernatant was collected, concentrated to 500 μl using an Amicon Ultra 15 filter (3 kDa cutoff membrane), and stored at -80°C before analysis. Cell numbers in each condition were determined to normalize the analyte (MCP-1, IL-6, IL-8, TGF-β1) concentration values ​​obtained using a Luminex®-based multiplex assay (R&D Systems) according to the manufacturer's instructions. All samples were analyzed in technical duplicates and performed on two different donors. Data were acquired on a MAGPIX reader (Millipore), and concentrations were derived from the measured mean fluorescence intensity (MFI) using a standard curve fitted using a five-parameter logistic regression (SSL5) using Milliplex Analyst software (Millipore).

[0086] In vitro multilineage differentiation

[0087] MSCs were seeded into 24-well plates and allowed to adhere overnight before removing the medium, which was then replaced with specific differentiation medium for osteogenesis and adipogenesis (STEMCELL Technologies) according to the manufacturer's protocol. Osteogenic induction was confirmed by Alizarin Red S staining (ScienCell) for calcium deposition, and adipogenic differentiation was confirmed by Oil Red O staining (Sigma) for lipid droplet detection. For chondrogenesis, MSCs were cultured at 1 × 10 in a 15 ml tube. 6 Cells were pelleted at 300 x g for 5 minutes per pellet, and chondrogenic medium (STEMCELL Technologies) was added to the pellet. The medium was changed every other day for 3 weeks before glycosaminoglycan staining with Safranin O.

[0088] Senescence assay

[0089] MSCs were seeded into 6-well plates at an initial cell seeding density of 1500 cells / cm. 2 Cells were seeded at 100°C and cultured for 3–5 days. Senescent cells were identified using a senescence β-galactosidase staining kit (Sigma-Aldrich) according to the manufacturer's protocol. Briefly, cells were fixed for 10 minutes at room temperature and then cultured overnight at 37°C in staining solution containing X-gal. Cells that stained positive with a blue precipitate, a result of X-gal substrate cleavage, indicated senescent cells.

[0090] Immunofluorescence and confocal microscopy

[0091] To detect SA-βgal in cells along with another senescence marker (γ-H2AX foci) (66) and the proliferation marker 5-ethynyl-2′-deoxyuridine (Edu), a protocol was performed as previously described (67). Briefly, cells were cultured with 10 μM Edu (Click-it® Edu Alexa Fluor® 488 Imaging Kit, Invitrogen) for 24 h, then fixed with 10% neutral-buffered formalin (Sigma) and stained for SA-βGal using the components of the staining kit according to the manufacturer's instructions (#9860, Cell Signaling Technology). Edu labeling was then detected according to the manufacturer's protocol (Invitrogen).

[0092] For immunostaining, cells were washed with PBS, incubated in blocking buffer (PBS containing 0.5% bovine serum albumin) for 1 hour, and then incubated with mouse anti-phospho-histone H2A.X (Ser139) antibody (05-636, Millipore) in blocking buffer overnight at 4°C. Next, cells were stained with Rhodamine Red. TMThe cells were incubated with βGal-conjugated secondary antibody (Jackson Immuno Research Laboratories) for 1 hour, washed three times with PBS, and counterstained with NucBlue (Hoechst 33342)-containing mounting solution (Invitrogen). Images were acquired with an FV1200 confocal microscope using a 20x objective (Olympus). Stained SA-βGal cells were also visualized by phase contrast microscopy.

[0093] The following references, many of which are cited herein, are incorporated by reference in their entireties:

[0094] References

[0095] [Table 2] TIFF0007825308000003.tif220170TIFF0007825308000004.tif218170TIFF0007825308000005.tif219170TIFF00078253080 00006.tif221170TIFF0007825308000007.tif221170TIFF0007825308000008.tif215170TIFF0007825308000009.tif232170

[0096] The details of one or more embodiments are set forth in the accompanying drawings and detailed description. Other features, objects, and advantages will become apparent from the detailed description, drawings, and claims. While a number of embodiments of the present invention have been described, it will be understood that various changes can be made without departing from the spirit and scope of the invention. It should also be understood that the accompanying drawings are not necessarily to scale and that they depict various features and underlying principles of the present invention in a somewhat simplified manner. The present application provides the following aspects of the invention. (Aspect 1) 1. A method for detecting senescent cells, comprising: loading a liquid sample containing a plurality of cells into the sensor; placing the sensor containing the liquid sample inside or near a detection coil of a magnetic resonance relaxometry device; and To detect the amount of senescent cells in the liquid sample, 2 measuring a value. (Aspect 2) The method of embodiment 1, wherein the sensor is a tube or a chamber. (Aspect 3) 2. The method of embodiment 1, wherein the amount of senescent cells in the liquid sample is determined relative to a reference sample. (Aspect 4) 2. The method of embodiment 1, wherein the amount of senescent cells in the liquid sample is proportional to the concentration of ferritin in the liquid sample. (Aspect 5) T to detect the amount of senescent cells 2 determining a value of Fe in the liquid sample; 3+ 2. The method of embodiment 1, comprising quantifying the amount of (Aspect 6) To determine whether senescent cells are present in the sample, 1 The relaxation time is T 2 2. The method of embodiment 1, wherein the method is used in combination with a value. (Aspect 7) Said T 1 or T 2 7. The method of embodiment 6, wherein determining the value comprises delivering a pulse train for a period of less than 1 minute. (Aspect 8) Said T 1 or T 2 7. The method of embodiment 6, wherein determining the value comprises acquiring and averaging between 2 and 70 scans. (Aspect 9) Said T 2 The method of embodiment 1, wherein the value decreases as the number of senescent cells increases. (Aspect 10) 2. The method of embodiment 1, wherein the cells comprise mesenchymal stromal cells. (Aspect 11) 2. The method of embodiment 1, wherein the cells comprise hematopoietic stem cells. (Aspect 12) 2. The method of embodiment 1, wherein the cells comprise induced pluripotent stem cells. (Aspect 13) 2. The method of embodiment 1, wherein the detection volume of the magnetic resonance relaxometry device comprises a sample volume of less than about 1 μL. (Aspect 14) T 2 2. The method of embodiment 1, wherein determining comprises measuring the paramagnetic or ferromagnetic ion content of the cells. (Aspect 15) 15. The method of embodiment 14, wherein the paramagnetic or ferromagnetic ions comprise copper. (Aspect 16) 1. A method for improving the availability of stem or progenitor cells to a patient, comprising: detecting senescent cells in a sample by the method of any one of aspects 1 to 15; enriching cells in the sample to reduce the number of senescent cells in the sample. (Aspect 17) 17. The method of embodiment 16, wherein enriching for cells comprises passing the sample through a cell separator, thereby separating senescent cells from non-senescent cells in the sample. (Aspect 18) 17. The method of embodiment 16, wherein the cell separation device comprises a microfluidic spiral path. (Aspect 19) 1. A system for detecting senescent cells, comprising: a magnetic resonance relaxometry device configured to detect the amount of senescent cells in a liquid sample; and the system comprising a cell separation device for reducing the amount of the senescent cells in the liquid sample based on output from the magnetic resonance relaxometry device. (Aspect 20) 20. The system of embodiment 19, wherein the liquid sample is contained within a microcapillary. (Aspect 21) 20. The system of embodiment 19, wherein the liquid sample is taken from a batch of cells. (Aspect 22) 22. The system of aspect 21, wherein the cell separation device removes senescent cells from the cell batch based on detecting the amount of the senescent cells. (Aspect 23) 20. The system of embodiment 19, wherein the cell separation device comprises a microfluidic cell sorter, an inertial focusing device, a microfluidic filtration device, a centrifugal flow device, a deterministic lateral displacement (DLD) chip, a tangential flow microfiltration device, or a combination thereof. (Aspect 24) 20. The system of embodiment 19, wherein the cell separation device comprises a microfluidic spiral path device. (Aspect 25) The magnetic resonance relaxometry device detects the amount of senescent cells in the liquid sample. 2 20. The system of embodiment 19, wherein the system is configured to measure a value. (Aspect 26) 20. The system of embodiment 19, wherein the amount of senescent cells in the liquid sample is proportional to the concentration of ferritin in the liquid sample. (Aspect 27) The magnetic resonance relaxometry device detects the amount of senescent cells. 2 and configured to measure Fe values ​​in the liquid sample. 3+ 20. The system of embodiment 19, comprising quantifying the amount of (Aspect 28) 20. The system of embodiment 19, wherein the senescent cells comprise mesenchymal stromal cells (MSCs), hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPSCs).

Claims

1. 1. A method for detecting senescent cells, comprising: loading a liquid sample containing a plurality of cells into a sensor; placing the sensor containing the liquid sample inside or near a detection coil of a magnetic resonance relaxometry device; and To detect the amount of senescent cells in the liquid sample, 2 measuring the value of The method, wherein (i) the amount of senescent cells in the liquid sample is proportional to the ferritin concentration in the liquid sample, or (ii) measuring the T2 value comprises quantifying the amount of Fe3+ in the sample.

2. The method of claim 1 , wherein the sensor is a tube or a chamber.

3. 10. The method of claim 1, wherein the amount of senescent cells in the liquid sample is measured relative to a reference sample.

4. To determine whether senescent cells are present in the sample, T 1 The relaxation time is T 2 The method of claim 1 , wherein the method is used in combination with a value.

5. Said T 1 or T 2 5. The method of claim 4, wherein measuring the value comprises applying a pulse train for a period of less than one minute.

6. Said T 1 or T 2 5. The method of claim 4, wherein measuring the value comprises acquiring and averaging between 2 and 70 scans.

7. Said T 2 The method of claim 1, wherein the value decreases as the number of senescent cells increases.

8. The method of claim 1 , wherein the cells comprise mesenchymal stromal cells.

9. The method of claim 1 , wherein the cells comprise hematopoietic stem cells.

10. The method of claim 1 , wherein the cells comprise induced pluripotent stem cells.

11. 10. The method of claim 1, wherein the detection volume of the magnetic resonance relaxometry device comprises a sample volume of less than about 1 μL.

12. T 2 10. The method of claim 1, wherein measuring the value comprises measuring the paramagnetic or ferromagnetic ion content of the cell.

13. The method of claim 12 , wherein the paramagnetic or ferromagnetic ions comprise copper.

14. 1. A method for improving stem or progenitor cell availability, comprising: Detecting senescent cells in a sample by the method of any one of claims 1 to 13; and enriching cells in the sample to reduce the number of senescent cells in the sample; The method is carried out in vitro.

15. 15. The method of claim 14, wherein enriching the cells comprises passing the sample through a cell separator, thereby separating senescent cells from non-senescent cells in the sample.

16. 16. The method of claim 15, wherein the cell separation device comprises a microfluidic spiral path.

17. 1. A system for detecting senescent cells, comprising: a magnetic resonance relaxometry device configured to detect the amount of senescent cells in a liquid sample; and a cell separation device for reducing the amount of senescent cells in the liquid sample based on an output from the magnetic resonance relaxometry device; The system, wherein (i) the amount of senescent cells in the liquid sample is proportional to the ferritin concentration in the liquid sample, or (ii) the magnetic resonance relaxometry device is configured to measure the T2 value to detect the amount of Fe3+ in the sample.

18. The system of claim 17 , wherein the liquid sample is contained within a microcapillary.

19. The system of claim 17 , wherein the liquid sample is taken from a batch of cells.

20. 20. The system of claim 19, wherein the cell separation device removes senescent cells from the cell batch based on detecting the amount of senescent cells.

21. 20. The system of claim 17, wherein the cell separation device comprises a microfluidic cell sorter, an inertial focusing device, a microfluidic filtration device, a centrifugal flow device, a deterministic lateral displacement (DLD) chip, a tangential flow microfiltration device, or a combination thereof.

22. 20. The system of claim 17, wherein the cell separation device comprises a microfluidic spiral path device.

23. The magnetic resonance relaxometry device detects the amount of senescent cells in the liquid sample. 2 20. The system of claim 17, configured to measure a value.

24. 18. The system of claim 17, wherein the senescent cells comprise mesenchymal stromal cells (MSCs), hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPSCs).

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